Neurotherapeutic composition and method therefor

ABSTRACT

Neurotherapeutically effective pharmaceutical compositions are described that include carboxypeptidase E inhibitors. One class of carboxypeptidase E inhibitors found to exhibit significant neurotropic activity are β-lactam compounds, particularly penam and cephem β-lactam antibiotics and non-antibiotic derivatives thereof.

This application is a continuation of U.S. patent application Ser. No.09/783,201, filed Feb. 14, 2001, which is expressly incorporated byreference herein.

FIELD OF INVENTION

This invention relates to a novel mechanism of neuropsychiatricintervention. More particularly, this invention is directed topharmaceutical formulations and methods for treatment of a variety ofneurological disease states, including cognitive and behavioraldisorders.

BACKGROUND AND SUMMARY OF THE INVENTION

The pharmaceutical industry has directed extensive research anddevelopment efforts toward discovery and commercialization of drugs fortreatment of neurological disorders. Such disorders typically derivefrom chemical imbalances in the brain. Overproduction or underproductionof pertinent neurochemical species and/or receptor dysfunction has beenidentified with many disease states recognized by neurologists,psychiatrists, psychologists and other medical practitioners skilled inthe diagnosis and treatment of mental disease. Most of the discoveryeffort for new neurologically active drugs has been based on the studyof agonist/antagonist drug interaction with one or more of the numerousreceptors in the brain and/or their respective receptor ligands.

The present invention provides a novel approach to drug intervention inthe treatment of a wide variety of neurologic disease states and otherdisease states or clinical conditions of related etiology. It is basedin part on the discovery that β-lactam containing compounds known fortheir activity as inhibitors of bacterial peptidases or proteases,particularly transpeptidases and/or carboxypeptidases, are alsoinhibitors of certain mammalian neuro-peptidases, includingN-acetylated-α-linked acidic peptidases (NAALADases), several of whichhave been identified/characterized in the literature [Pangalos et al.,J. Biol. Chem., 1999, 274 No. 13, 8470-8783]. The present invention isalso based in part on the discovery that neurogenic carboxypeptidasescan be targeted with carboxypeptidase inhibitors to effect significantbehavioral modification and enhanced cognitive performance. Preliminarystudies have confirmed that one or more neurogenic proteases, nowbelieved to be NAALADases and related peptidases and transferases,capable of recognizing and transforming certain neuropeptides (e.g.,N-acetyl-L-aspartyl-L-glutamate) play a significant if not dominant roleat the neurochemical level of brain function and concomitantly have asubstantial impact on patient behavior and cognitive performance. It hasbeen previously reported that certain glutamate analogs acting asNAALADase inhibitors can be used to treat prostate disease and glutamateabnormalities associated with certain nervous tissue insult. It has nowbeen determined that certain β-lactam-containing bacterial peptidaseinhibitors capable of blood-brain barrier transport, can function in thebrain at very low concentrations as potent neuroactive drug substancesto reduce the symptoms of a wide variety of neurological disorderscharacterized by behavioral aberration or sensory/cognitive dysfunction.Significantly, such bacterial enzyme inhibitors are believed to beeffective inhibitors of neurogenic peptidases, particularlycarboxypeptidase E, at concentrations below those concentrations knownto be required for clinically effective bacterial enzyme inhibition.

Accordingly, one embodiment of the present invention is directed to amethod for treatment of cognitive and behavioral disorders inwarm-blooded vertebrates by administering compounds known for theiractivity as bacterial protease or peptidase inhibitors, which compounds,when present at effective concentrations in the brain, have now beendetermined to be capable of inhibiting or otherwise modulating theactivity of one or more neurogenic enzymes.

In a related embodiment there is provided method for treatment ofcognitive and behavioral disorders in a patient in need of suchtreatment. The method comprises the step of inhibiting neurogenicpeptidases, particularly, carboxypeptidase E and related neurogenicenzymes. Such neuropeptidase inhibition is effected by administering aneffective amount of a β-lactam compound recognized for its capacity tobind to and inhibit a bacterial enzyme, for example, a β-lactamase or abacterial protease involved in bacterial cell wall synthesis. Suchbacterial proteases are known in the art as “penicillin bindingproteins.” Exemplary of β-lactam compounds for use in this invention aremoxalactam, its salts, esters and structurally related cephems and1-oxa-1-dethia cephems. Effective inhibition ofneurogenic-carboxypeptidase E and related neuro-peptidase activity inwarm-blooded vertebrates in accordance with this invention has beenfound to produce marked enhancement in cognitive performance andbehavioral management.

Exemplary of cognitive and behavioral disorders susceptible to treatmentin accordance with this invention include aggressive disorder, obsessivecompulsive disorder, anxiety, depression, ADHD, and memory impairment.Animal data suggest that the method and formulation of this inventionhave potential as an antiaggressive agent to control impulsivity andviolence in autism, Tourette's syndrome, mental retardation, psychosis,mania, senile dementia and individuals with personality disorders andhistory of inappropriate aggression. Clinic applications extend to thetreatment of children with ADHD and conduct disorder, as an anxiolytic,and as a cognition enhancer for the geriatric population to improvelearning and memory and to ameliorate disorientation.

In another embodiment of this invention there is provided a method oftreating a patient afflicted with a condition, or disposed todevelopment of a condition, characterized at least in part by abnormalextracellular concentration of glutamate in the brain or other nervoustissue. The method comprises the step of administering to the patient ineffective amounts of a compound capable of inhibiting the activity of apenicillin-binding protein of bacterial origin. The composition isadministered in an amount effective to prevent or alleviate the symptomsof such condition. The method and formulation embodiments of theinvention find use in both human health and veterinary applications,e.g., in canine, feline and equine species.

In one embodiment of the present invention a warm-blooded vertebrate,most typically a human patient, affected by a neurologic disease statecharacterized by cognitive or behavioral abnormalities is treated with a1-oxa-1-dethia cephalosporin, more preferably a 7-methoxy-1-oxa-1-dethiacephalosporin, optionally as an active ester derivative in an orally(including buccal or sublingual administration) or a parenterallyadministered formulation. In one embodiment, the peptidase inhibitor ismoxalactam,[7-β-[2-carboxy-2-(4-hydroxyphenyl)acetamido]-7α-methoxy-3-[[(1-methyl-1H-tetrazol-5-yl)thio]methyl]-1-oxa-1-dethia-3-cephem-4-carboxylicacid], described and claimed with related compounds, including theirorally absorbed active ester derivatives, in U.S. Pat. No. 4,323,567,the specification of which is expressly incorporated herein byreference. Moxalactam has been found to exhibit significant doseresponsive neuroactivity when administered parenterally at least atabout 50 μg/kg of body weight.

In another embodiment of the present invention there is provided apharmaceutical formulation for treatment with consequent reduction ofsymptoms of behavioral or cognitive disorders in patients in need ofsuch treatment. The formulation comprises a compound characterized notonly by its affinity to bacteria derived penicillin-binding proteins,but as well, its affinity to neurogenic carboxypeptidases, particularlycarboxypeptidase E. In that embodiment the level of activity exhibitedby the carboxypeptidase inhibitor in the present method is not onlydependent on its affinity to penicillin-binding proteins and tocarboxypeptidase, namely carboxypeptidase-E, it is also particularlydependent on ability of the inhibitor compound to cross the blood brainbarrier to achieve levels in the brain effective to modify patientbehavior and/or cognitive performance. While the formulations of thisinvention can be prepared specifically for any art-recognized mode ofadministration capable of achieving threshold minimum proteaseinhibiting concentrations in the brain, they are typically formulatedfor parenteral or oral administration, optionally in the form ofprolonged release or “drug depot” type formulations well known in theart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-42 are graphic representations of data gathered in the conductof testing of moxalactam, other β-lactam antibiotics, clavulanic acidand other neuroactive compounds in various animal models accepted in theart for detection of activity against offensive aggression (FIGS. 1-4,9-14, 24, 29, 31 and 32), general motor activity, olfactorydiscrimination (FIG. 5), sexual activity (FIG. 6), anxiolytic activity(FIGS. 7, 25, 26, 28, 37 and 40), and spatial memory (FIGS. 8 and29-36). FIGS. 15 and 16 are graphic illustrations of the effect ofintracerebrally administered peptidoglycan-precursor protein onoffensive aggression and olfactory discrimination in hamsters.

DETAILED DESCRIPTION OF THE INVENTION

The present invention and the various embodiments described and claimedherein derive, in part, from the discoveries that compounds capable ofbinding to and inhibiting enzyme activity of penicillin-binding proteinsof bacterial origin are also potent inhibitors of N-acetylated-α-linkedacid dipeptidase (NAALADase) activity and other related enzymes in thebrain, including carboxypeptidase E, and that when administered toprovide effective threshold enzyme inhibitory concentrations of same inthe brain, such inhibitors exhibit clinically significant neuroactivityevidenced in part by behavioral modification and enhanced cognition andfunction.

In one embodiment the peptidase inhibitors effective for use inaccordance with the present invention are characterized by theircapacity to inhibit a bacterial protease exhibiting selectiveproteolytic activity on a protein or peptide substrate comprisingacyl-D-alanyl-D-alanine. Alternatively stated, effective inhibitors foruse in treatment of behavioral and cognitive disorders in accordancewith one embodiment of this invention, can be characterized by theirselective affinity (by associative and/or covalent binding) topenicillin-binding proteins; such compounds include particularlyβ-lactam antibiotics such as penicillins, cephalosporins and analoguesthereof, particularly 1-oxa-1-dithiacephems. Based on animal tests todate, such bacterial protease inhibitors appear to function atsubclinical-antibiotic levels in the brain to inhibit neuropeptidaseactivity which can function in neurochemical mediation of behavior andcognitive performance. Effective inhibition of neuropeptidase activitywith concomitant mediation of behavior and cognitive performance hasalso been effected by administration of a β-lactamase inhibitor,clavulanic acid, a β-lactam containing compound having no clinicallysignificant antibiotic activity. It is surmised that inhibition ofneuropeptidase activity allows modulation of the concentration and/orfunction of one or more neurotransmitters or neuromodulators withconcomitant improvement in neurological function evidenced byenhancement of cognitive performance and attenuation of aberrantbehavioral phenotypes.

In one example of this invention, moxalactam given i.p. at 50micrograms/kg inhibits aggression in hamsters, enhances spatial learningin rats, and acts as an anxiolytic in rats. Clavulanic acid has shownanxiolytic activity when administered i.p. at less than 1 microgram/kg;it also exhibits potent neuroprotectant activity, but animal testingdata available for clavulanic acid to date do not evidence the potentantiaggression and cognition enhancement activity exhibited bymoxalactam (although clavulanic acid did exhibit a modest level of suchactivities). Nor does moxalactam appear to exhibit the neuroprotectantactivity seen for clavulanic acid. The unique neurological activityprofiles of clavulanic acid and moxalactam provides strong evidence thatthe compounds are each interacting with unique sets of neurogenictargets.

Historically, those knowledgeable in the field of beta lactamantibiotics understand that the mode of action as antibacterial agentsis by inhibiting cell wall synthesis by acting as a substrate forpenicillin-binding protein (PBP); the term PBP has been extended toinclude binding to all beta lactams including cephalosporins. Morerecently, investigators have been able to clone and sequence these PBP'sas well as crystallize the enzymes and determine active site motifs (seeP. Palomeque et al., J. Biochem., 279, 223-230, 1991). Based on thisdata, the four putative binding sites for PBP have been designated sitesI, II, III and IV. The sites, sequence location and amino acid (AA)sequence are as follows:

Site I: 35 AA's downstream from N-terminus: STTK (SEQ ID NO: 1) Site II:57 AA's downstream from STTK (SEQ ID NO: 1) motif: SGC, SGN, or SAN SiteIII: 111 AA's downstream from SGC motif: KTG Site IV: 41 AA's downstreamfrom SGC motif: ENKD (SEQ ID NO: 2)

Pursuant to identifying an enzyme system in the brain that moxalactamwould inhibit in a similar manner to PBP, it was discovered that aglutamyl carboxypeptidase enzyme known as N-acetyl-α-linked acidicdipeptidase (NAALADase) (See M. N. Pangalos et al., J. Bio. Chem., 264,8470-8483, 1999) has an almost perfect overlap of the putative activesites of PBP. This enzyme system is responsible for regulating theglutamatergic neurotransmission pathways, the effects of which would beexpressed in such behavioral outcomes as aggression, memory/cognition,and anxiety. As a result of the almost perfect overlap of the putativeactive sites of PBP and the conserved sequences in human and ratNAALADase, it was initially believed that moxalactam and other β-lactamcompounds mediate behavioral effects by inhibiting NAALADase at lowconcentrations. This was based on the following overlap sequencesimilarity between PBP and NAALADase I, one of several known NAALADasespecies, as shown below:

Site I: PBP: 35 AA's downstream from N-terminus: STTK (SEQ ID NO:1)NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO: 3) SiteII: PBP: 57 AA's downstream from STTK (SEQ ID NO: 1) motif: SGC, SGN, orSAN NAALADase: 59 AA's downstream from STQK (SEQ ID NO: 3) motif: SFGSite III: PBP: 111 AA's downstream from SGC motif: KTG NAALADase: 110AA's downstream from SFG motif: KLG Site IV: PBP: 41 AA's downstreamfrom SGC motif: ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream fromSFG motif: ERGV (SEQ ID NO: 4)

Since the beta-lactams provide their inhibition of PBP transpeptidationof bacterial cell wall by binding to these four active sites, one caninfer that the conserved similarity in active site sequences andlocation would confer similar binding properties of moxalactam and otherβ-lactam compounds to NAALADase and possibly other neurogenic enzymeshaving sequences overlapping with the four active binding site motif.Recent computer modeling experiments have shown that while clavulanicacid exhibits a good fit with NAALADase, moxalactam does not, suggestinganother neurogenic target for moxalactam. Further molecular modelingstudies have suggested that the neurogenic target for moxalactam isanother neurogenic peptidase, carboxypeptidase E. That discovery coupledwith observation of the significant behavioral modification effectsderiving from administration of very low doses of certain penicillinprotein binding compounds has provided insight into a novel approach tothe prevention and treatment of disease states characterized byneurological dysfunction.

The unique neurological activity profiles of the two β-lactam compoundsthat have been studied most extensively to date, moxalactam andclavulanic acid, suggest that those compounds exhibit activity onmultiple neurogenic enzyme targets, including NAALADase and structurallyrelated enzymes, particularly those that might share the four activebinding site motif common to both PBP and NAALADase. To identify otherputative neurogenic targets for the behavioral and cognitive activitiesdiscovered for moxalactam and clavulanic acid, the sequence forNAALADase II was used to search the human genome database (NCBI-BLAST).Seven human gene sequences were identified that have significanthomology with NAALADase H and that encode for the four active sitemotif:

1) >dbj/AP001769.2/AP001769 Homo sapiens chromosome 11 clone RP11-240F8map 11q14 Site I: PBP: 35 AA's downstream from N-terminus . . . STTK(SEQ ID NO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQID NO: 3) >dbj/AP001769: NSRK (SEQ ID NO: 5) Site II: PBP: 57 AA'sdownstream from STTK (SEQ ID NO: 1) motif: SGC, SGN, or SAN NAALADase:59 AA's downstream from STQK (SEQ ID NO:3) motif: SFG >dbj/AP001769: SFGSite III PBP: 111 AA's downstream from SGC motif . . . KTG NAALADase:110 AA's downstream from SFG motif: KLG >dbj/AP001769: KLG Site IV: PBP:41 AA's downstream from SGC motif . . . ENKD (SEQ ID NO: 2) NAALADase:41 AA's downstream from SFG motif: ERGV (SEQ ID NO: 4) >dbj/AP001769:ERSI (SEQ ID NO: 6) 2) >dbj|AP000827.2|AP000827 Homo sapiens chromosome11 clone RP. Site I: PBP: 35 AA's downstream from N-terminus . . . STTK(SEQ ID NO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQID NO: 3) >dbj|AP000827.2: NSRK (SEQ ID NO: 5) Site II: PBP: 57 AA'sdownstream from STTK (SEQ ID NO: 1) motif: . . . SGC, SGN, or SANNAALADase: 59 AA's downstream from STQK (SEQ ID NO: 3) motif:SFG >dbj|AP000827.2: SFG Site III: PBP:111 AA's downstream from SGCmotif . . . KTG NAALADase: 110 AA's downstream from SFG motif:KLG >dbj|AP000827.2: KLG Site IV: PBP: 41 AA's downstream from SGC motif. . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream from SFG motif:ERGV (SEQ ID NO: 4) >dbj|AP000827.2: ERSI (SEQ ID NO: 6)3) >dbj|AP000648.2|AP000648 Homo sapiens chromosome 11 clone CM. Site I:PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID NO: 1)NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO:3) >>dbj∩AP000648.2: NSRK (SEQ ID NO: 5) Site II: PBP: 57 AA'sdownstream from STTK (SEQ ID NO: 1) motif . . . SGC, SGN, or SANNAALADase: 59 AA's downstream from STQK (SEQ ID NO: 3) motif:SFG >dbj|AP000648.2: SFG Site III PBP:111 AA's downstream from SGC motif. . . KTG NAALADase: 110 AA's downstream from SFG motif:KLG >dbj|AP000648.2: KLG Site IV: PBP: 41 AA's downstream from SGC motif. . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream from SFG motif:ERGV (SEQ ID NO: 4) >dbj|AP000648.2: ERSI (SEQ ID NO: 6)4) >gb|AC074003.2|AC074003 Homo sapiens chromosome 2 clone RP11. Site I:PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID NO: 1)NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO: 3)gb|AC074003.2|AC074003: STQ- Site II: PBP: 57 AA's downstream from STTK(SEQ ID NO: 1) motif: . . . SGC, SGN, or SAN NAALADase: 59 AA'sdownstream from STQK (SEQ ID NO: 3) motif: SFG gb|AC074003.2|AC074003:SFG Site III: PBP: 111 AA's downstream from SGC motif . . . KTGNAALADase: 110 AA's downstream from SFG motif: KLGgb|AC074003.2|AC074003: KLG Site IV: PBP: 41 AA's downstream from SGCmotif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream from SFGmotif: ERGV (SEQ ID NO: 4) gb|AC074003.2|AC074003 ERGV (SEQ ID NO: 4)5) >emb|AL162372.6|AL162372 Homo sapiens chromosome 13 clone RP. Site I:PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID NO: 1)NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO: 3)emb|AL162372.6: STQ- Site II: PBP: 57 AA's downstream from STTK (SEQ IDNO: 1) motif . . . SGC, SGN, or SAN NAALADase: 59 AA's downstream fromSTQK (SEQ ID NO: 3) motif: SFG emb|AL162372.6: SFG Site II: PBP: 111AA's downstream from SGC motif . . . KTG NAALADase: 110 AA's downstreamfrom SFG motif: KLG emb|AL162372.6: KLG Site IV: PBP: 41 AA's downstreamfrom SGC motif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstreamfrom SFG motif: ERGV (SEQ ID NO: 4) emb|AL162372.6 ERGV (SEQ ID NO: 4)6) gb|AC024234.5|AC024234 Homo sapiens chromosome 11 clone RP1. Site I:PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID NO: 1)NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO: 3)gb|AC024234.5|AC024234: STQ- Site II: PBP: 57 AA's downstream from STTK(SEQ ID NO: 1) motif: . . . SGC, SGN, or SAN NAALADase: 59 AA'sdownstream from STQK (SEQ ID NO: 3) motif: SFG gb|AC024234.5|AC024234:SFG Site III: PBP: 111 AA's downstream from SGC motif . . . KTGNAALADase: 110 AA's downstream from SFG motif KLGgb|AC024234.5|AC024234: KLG Site IV: PBP: 41 AA's downstream from SGCmotif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream from SFGmotif: ERGV (SEQ ID NO: 4) gb|AC024234.5|AC024234 ERGV (SEQ ID NO: 4) 7)dbj|AP002369.1|AP002369 Homo sapiens chromosome 11 clone RP . . . SiteI: PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID NO: 1)NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO: 3)dbj|AP002369.1: STQ- Site II: PBP: 57 AA's downstream from STTK (SEQ IDNO: 1) motif: . . . SGC, SGN, or SAN NAALADase: 59 AA's downstream fromSTQK (SEQ ID NO: 3) motif: SFG dbj|AP002369.1: SFG Site III: PBP: 111AA's downstream from SGC motif . . . KTG NAALADase: 110 AA's downstreamfrom SFG motif: KLG dbj|AP002369.1: KLG Site IV: PBP: 41 AA's downstreamfrom SGC motif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstreamfrom SFG motif: ERGV (SEQ ID NO: 4) dbj|AP002369.1 ERGV (SEQ ID NO: 4)

The encoded protein of each of those gene sequences expressed in thebrain are probable targets for behavioral and cognitive activity byβ-lactams and other NAALADase inhibitors. Thus in accordance with oneaspect of this invention there is provided a method for modifyingbehavior and/or cognition comprising the step of inhibiting thebiological activity of the non-NAALADase protein(s) expressed by one ormore of the above-identified gene sequences, by administering aneffective amount of a β-lactam compound or other compound capable ofpeptidase inhibition. As stated above, recent molecular modeling studiesnow suggest that carboxypeptidase E is the enzyme which is inhibited bymoxalactam in neural tissues to provide basis for a multiplicity ofneurotherapeutic effects.

In one embodiment the peptidase inhibitors effective for use in thevarious pharmaceutical formulation and method embodiments of thisinvention, generally speaking, are compounds which exhibit detectableselective affinity for art recognized penicillin-binding proteins,including particularly β-lactam-containing compounds (hereinafter“β-lactam compounds”) such as penicillins and cephalosporins, and mostpreferably, ceratin 1-oxa-1-dithiacephem analogues thereof, certainβ-lactamase inhibitors, and peptides comprising the amino acid sequenceAla-D-γ-Glu-Lys-D-Ala-D-Ala. Among such peptidase inhibiting compounds,those preferred for use in accordance with this invention are compoundsthat also exhibit good blood brain barrier transport propertiesevidenced by favorable cerebral spinal fluid (CSF)/brain:serumconcentration ratios. Further, it will be appreciated that otherart-recognized peptidase inhibitors may be used alone or in combinationwith penicillin protein-binding compounds for treatment and preventionof behavioral and/or cognitive disorders.

In the embodiments of the invention directed to pharmaceuticalformulations for use in inhibition of neurogenic peptidase to modifybehavior and/or improve cognitive function, the β-lactam compounds aretypically formulated in unit dosage form optionally in combination with,or as covalent conjugates of, other compounds or molecular entities,respectively, known to enhance drug transport across the blood brainbarrier. Such drug formulation/conjugation techniques are described andclaimed in the following listed United States Patents: U.S. Pat. Nos.5,624,894; 5,672,683; 5,525,727; 5,413,996; 5,296,483; 5,187,158;5,177,064; 5,082,853; 5,008,257; 4,933,438; 4,900,837; 4,880,921;4,824,850; 4,771,059; and 4,540,564.

Enhanced concentrations of drug substances in the brain can also beachieved by co-administration with P-glycoprotein efflux inhibitors suchas those described in U.S. Pat. Nos. 5,889,007; 5,874,434; 5,654,304;5,620,855; 5,643,909; and 5,591,715. Alternatively, β-lactam antibioticcompounds useful in accordance with this invention, includingparticularly 1-oxa-1-dethia cephems, can be administered alone or incombination with art-recognized β-lactamase inhibitors, which themselvesmay or may not be β-lactam compounds or compounds capable of exhibitingselective affinity for penicillin-binding proteins. Examples ofβ-lactamase inhibitors which can be used in combination with otherneuropeptidase inhibitors useful in accordance with this invention fortreatment and/or prevention of cognitive or behavioral disorders areother β-lactam compounds which may or may not exhibit independentclinically significant antibacterial activity, such as clavulanic acidand thienamycin and analogs thereof, sulbactam, tazobactam,sultamicillin, and aztreonam and other monolactams.

The patent and non-patent literature is replete with referencesdescribing β-lactam antibiotics, their preparation, theircharacterization, their formulation and their mode of action. β-Lactamantibiotics are known to exhibit their antibiotic activity byinterfering with one or more biological pathways involved in bacteriacell wall synthesis; more particularly, they inhibit carboxypeptidaseand/or transpeptidase (or protease) activity involved in cross-linkingof the peptidoglycan chains used as building blocks for cell wallsynthesis. β-Lactam antibiotics are thus believed to act as inhibitorsof carboxypeptidases or transpeptidases by their covalent, and by somereports, noncovalent associative bonding, to one or more of a group ofsuch bacterial enzymes generally termed penicillin binding proteins(PBP's). Such enzymes serve to complete bacteria cell wall synthesis bycross linking peptidoglycan chains.

A similar peptidase-substrate interaction/inhibition is now suggested inaccordance with this invention as a significant neurochemical pathwayinvolved in brain function pivotal to cognitive performance andbehavioral phenotype. Such a neurochemical mechanism is suggested too bythe discovery that delivery of effective amounts of the peptideAla-D-γ-Glu-Lys-D-alanyl-D-alanine directly into the brain produced thesame modified behavioral characteristics as that achieved by comparableconcentrations of β-lactam compounds in the brain. The peptide appearsto serve as a substitute substrate for (and thus serve to inhibit theactivity thereof) one or more neurogenic peptidases (e.g., NAALADases)that normally exhibit their activity on peptidic neurotransmitters orneuromodulators, i.e., NAAD, in the ordinary course of certainneurochemical processes that mediate cognitive performance andbehavioral phenotype.

Based on animal tests to date it is believed that the general classes ofbehavioral disorders can be prevented or treated in accordance with thisinvention by administration of effective amounts of inhibitors of otherneurogenic peptidases, include aggressive disorder, obsessive-compulsivedisorder, anxiety, depression, and attention deficient hyperactivitydisease (ADHD). Thus in one embodiment of the invention acarboxypeptidase inhibitor, more specifically an inhibitor ofcarboxypeptidase E, is administered as an anti-aggressive agent tocontrol impulsivity and violence in a patient afflicted with autism,Tourette's Syndrome, mental retardation, psychosis, mania, seniledementia or that in a patient with personality disorder and history ofinappropriate aggression.

Other neurological disease states which can be treated in accordancewith the present invention include depression, including majordepression (single episode, recurrent, melancholic), atypical, dysthmia,subsyndromal, agitated, retarded, co-morbid with cancer, diabetes, orpost-myocardial infarction, involutional, bipolar disorder, psychoticdepression, endogenous and reactive, obsessive-compulsive disorder, orbulimia. In addition, peptidase inhibitors can be used to treat patientssuffering from pain (given alone or in combination with morphine,codeine, or dextroproposyphene), obsessive-compulsive personalitydisorder, post-traumatic stress disorder, hypertension, atherosclerosis,anxiety, anorexia nervosa, panic, social phobia, stuttering, sleepdisorders, chronic fatigue, cognition deficit associated withAlzheimer's disease, alcohol abuse, appetite disorders, weight loss,agoraphobia, improving memory, amnesia, smoking cessation, nicotinewithdrawal syndrome symptoms, disturbances of mood and/or appetiteassociated with pre-menstrual syndrome, depressed mood and/orcarbohydrate craving associated with pre-menstrual syndrome,disturbances of mood, disturbances of appetite or disturbances whichcontribute to recidivism associated with nicotine withdrawal, circadianrhythm disorder, borderline personality disorder, hypochondriasis,pre-menstrual syndrome (PMS), late luteal phase dysphoric disorder,pre-menstrual dysphoric disorder, trichotillomania, symptoms followingdiscontinuation of other antidepressants, aggressive/intermittentexplosive disorder, compulsive gambling, compulsive spending, compulsivesex, psychoactive substance use disorder, sexual disorder,schizophrenia, premature ejaculation, or psychiatric symptoms selectedfrom stress, worry, anger, rejection sensitivity, and lack of mental orphysical energy.

Other examples of pathologic, psychologic conditions which may betreated in accordance with this invention include, but are not limitedto: Moderate Mental Retardation (318.00), Severe Mental Retardation(318.10), Profound Mental Retardation (318.20), Unspecified MentalRetardation (319.00), Autistic Disorder (299.00), Pervasive DevelopmentDisorder NOS (299.80), Attention-Deficit Hyperactivity Disorder(314.01), Conduct Disorder, Group Type (312.20), Conduct Disorder,Solitary Aggressive Type (312.00), Conduct Disorder, UndifferentiatedType (312.90), Tourette's Disorder (307.23), Chronic Motor or Vocal TicDisorder (307.22), Transient Tic Disorder (307.21), Tic Disorder NOS(307.20), Primary Degenerative Dementia of the Alzheimer Type, SenileOnset, Uncomplicated (290.00), Primary Degenerative Dementia of TheAlzheimer Type, Senile Onset, with Delirium (290.30), PrimaryDegenerative Dementia of the Alzheimer Type, Senile Onset, withDelusions (390.20), Primary Degenerative Dementia of the Alzheimer Type,Senile Onset, with Depression (290.21), Primary Degenerative Dementia ofthe Alzheimer Type, Presenile Onset, Uncomplicated (290.10), PrimaryDegenerative Dementia of the Alzheimer Type, Presenile Onset, withDelirium (290.11), Primary Degenerative Dementia of the Alzheimer Type,Presenile Onset, with Delusions (290.12), Primary Degenerative Dementiaof the Alzheimer Type, Presenile Onset, with Depression (290.13),Multi-infarct dementia, Uncomplicated (290.40), Multi-infarct dementia,with Delirium (290.41), Multi-infarct Dementia, with Delusions (290.42),Multi-infarct Dementia, with Depression (290.4 3), Senile Dementia NOS(290.10), Presenile Dementia NOS (290.10), Alcohol Withdrawal Delirium(291.00), Alcohol Hallucinosis (291.30), Alcohol Dementia Associatedwith Alcoholism (291.20), Amphetamine or Similarly ActingSympathomimetic Intoxication (305.70), Amphetamine or Similarly ActingSympathomimetic Delusional Disorder (292.11), Cannabis DelusionalDisorder (292.11), Cocaine Intoxication (305.60), Cocaine Delirium(292.81), Cocaine Delusional Disorder (292.11), HallucinogenHallucinosis (305.30), Hallucinogen Delusional Disorder (292.11),Hallucinogen Mood Disorder (292.84), Hallucinogen PosthallucinogenPerception Disorder (292.89), Phencyclidine (PCP or Similarly ActingArylcyclohexylamine Intoxication (305.90), Phencyclidine (PCP) orSimilarly Acting Arylcyclohexylamine Delirium (292.81), Phencyclidine(PCP) or Similarly Acting Arylcyclohexylamine Delusional Disorder(292.11), Phencyclidine (PCP) or Similarly Acting ArylcyclohexylamineHood Disorder (292.84), Phencyclidine (PCP) or Similarly ActingArylcyclohexylamine Organic Mental Disorder NOS (292.90), Other orunspecified Psychoactive Substance Intoxication (305.90), Other orUnspecified Psychoactive Substance Delirium (292.81), Other orUnspecified Psychoactive Substance Dementia (292.82), Other orUnspecified Psychoactive Substance Delusional Disorder (292.11), Otheror Unspecified Psychoactive Substance Hallucinosis (292.12), Other orUnspecified Psychoactive Substance Mood Disorder (292.84), Other orUnspecified Psychoactive Substance Anxiety Disorder (292.89), Other orUnspecified Psychoactive Substance Personality Disorder (292.89), Otheror Unspecified Psychoactive Substance Organic Mental Disorder NOS(292.90), Delirium (293.00), Dementia (294.10), Organic DelusionalDisorder (293.81), Organic Hallucinosis (293.81), Organic Mood Disorder(293.83), Organic Anxiety Disorder (294.80), Organic PersonalityDisorder (310.10), Organic Mental Disorder (29.80), Obsessive CompulsiveDisorder (300.30), Post-traumatic Stress Disorder (309.89), GeneralizedAnxiety Disorder (300.02), Anxiety Disorder NOS (300.00), BodyDysmorphic Disorder (300.70), Hypochondriasis (or HypochondriacalNeurosis) (300.70), Somatization Disorder (300.81), UndifferentiatedSomatofoiin Disorder (300.70), Somatoform Disorder NOS (300.70),Intermittent Explosive Disorder (312.34), Kleptomania (312.32),Pathological Gambling (312.31), Pyromania (312.33), Trichotillomania(312.39), and Impulse Control Disorder NOS (312.39).

Additional examples of pathologic psychological conditions which may betreated using β-lactam containing peptidase inhibitors as described inthis invention include Schizophrenia, Catatonic, Subchronic, (295.21),Schizophrenia, Catatonic, Chronic (295.22), Schizophrenia, Catatonic,Subchronic with Acute Exacerbation (295.23), Schizophrenia, Catatonic,Chronic with Acute Exacerbation (295.24), Schizophrenia, Catatonic, inRemission (295.55), Schizophrenia, Catatonic, Unspecified (295.20),Schizophrenia, Disorganized, Chronic (295.12), Schizophrenia,Disorganized, Subchronic with Acute Exacerbation (29 5.13),Schizophrenia, Disorganized, Chronic with Acute Exacerbation (295.14),Schizophrenia, Disorganized, in Remission (295.15), Schizophrenia,Disorganized, Unspecified (295.10), Schizophrenia, Paranoid, Subchronic295.31), Schizophrenia, Paranoid, Chronic (295.32), Schizophrenia,Paranoid, Subchronic with Acute Exacerbation (295.33), Schizophrenia,Paranoid, Chronic with Acute Exacerbation (295.34), Schizophrenia,Paranoid, in Remission (295.35), Schizophrenia, Paranoid, Unspecified(295.30), Schizophrenia, Undifferentiated, Subchronic (295.91),Schizophrenia, Undifferentiated, Chronic (295.92), Schizophrenia,Undifferentiated, Subchronic with Acute Exacerbation (295.93),Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation(295.94), Schizophrenia, Undifferentiated, in Remission (295.95),Schizophrenia, Undifferentiated, Unspecified (295.90), Schizophrenia,Residual, Subchronic (295.61), Schizophrenia, Residual, Chronic(295.62), Schizophrenia, Residual, Subchronic with Acute Exacerbation(295.63), Schizophrenia, Residual, Chronic with Acute Exacerbation(295.94), Schizophrenia, Residual, in Remission (295.65), Schizophrenia,Residual, unspecified (295.60), Delusional (Paranoid) Disorder (297.10),Brief Reactive Psychosis (298.80), Schizophreniform Disorder (295.40),Schizoaffective Disorder (295.70), induced Psychotic Disorder (297.30),Psychotic Disorder NOS (Atypical Psychosis) (298.90), Bipolar Disorder,Mixed, Severe, without Psychotic Features (296.63), Bipolar Disorder,Manic, Severe, without Psychotic Features (296.43), Bipolar Disorder,Depressed, Severe, without Psychotic Features (296.53), BipolarDisorder, Mixed, with Psychotic Features (296.64), Bipolar Disorder,Manic, with Psychotic Features (296.44), Bipolar Disorder, Depressed,with Psychotic Features (296.54), Bipolar Disorder NOS (296.70), MajorDepression, Single Episode, with Psychotic Features (296.24), MajorDepression, Recurrent with Psychotic Features (296.34) PersonalityDisorders, Paranoid (301.00), Personality Disorders, Schizoid (301.20),Personality Disorders, Schizotypal (301.22), Personality Disorders,Antisocial (301.70), Personality Disorders, Borderline (301.83).

Anxiety disorders which may be treated in accordance with this inventioninclude, but are not limited to, Anxiety Disorders (235), Panic Disorder(235), Panic Disorder with Agoraphobia (300.21), Panic Disorder withoutAgoraphobia (300.01), Agoraphobia without History of Panic Disorders(300.22), Social Phobia (300.23), Simple Phobia (300.29), OrganicAnxiety Disorder (294.80), Psychoactive Substance Anxiety Disorder(292.89), Separation Anxiety Disorder (309.21), Avoidant Disorder ofChildhood or Adolescence (313.21), and Overanxious Disorder (313.00).

Effective amounts of the β-lactam carboxypeptidase inhibiting compoundsdescribed herein, can be used for the treatment of the followingpathologic psychological conditions: Moderate Mental Retardation; SevereMental Retardation; Profound Mental Retardation; Autistic Disorder;Attention-Deficit Hyperactivity Disorder; Pervasive Development DisorderNOS; Conduct Disorder, Group Type; Conduct Disorder, Solitary AggressiveType; Tourette's Disorder; Primary Degenerative Dementia of theAlzheimer Type, Senile Onset, with Delirium; Primary DegenerativeDementia of the Alzheimer Type, Senile Onset, with Delusions; PrimaryDegenerative Dementia of the Alzheimer Type, Presenile Onset;Schizophrenia, Catatonic, Subchronic; Schizophrenia, Catatonic, Chronic;Schizophrenia, Catatonic, Subchronic with Acute Exacerbation;Schizophrenia, Catatonic, Chronic with Acute Exacerbation;Schizophrenia, Catatonic, in Remission; Schizophrenia, Catatonic,Unspecified; Schizophrenia, Disorganized, Subchronic; Schizophrenia,Disorganized, Chronic; Schizophrenia, Disorganized, Subchronic withAcute Exacerbation; Schizophrenia, Disorganized, Chronic with AcuteExacerbation; Schizophrenia, Disorganized, in Remission; Schizophrenia,Disorganized, Unspecified; Schizophrenia, Paranoid, Subchronic;Schizophrenia, Paranoid, Chronic; Schizophrenia, Paranoid, Subchronicwith Acute Exacerbation; Schizophrenia, Paranoid, Chronic with AcuteExacerbation; Schizophrenia, Paranoid, in Remission; Schizophrenia,Paranoid, Unspecified; Schizophrenia, Undifferentiated, Subchronic;Schizophrenia, Undifferentiated, Chronic; Schizophrenia,Undifferentiated, Subchronic with Acute Exacerbation; Schizophrenia,Undifferentiated, Chronic with Acute Exacerbation; Schizophrenia,Undifferentiated, in Remission; Schizophrenia, Undifferentiated,Unspecified; Schizophrenia, Residual, Subchronic; Schizophrenia,Residual Chronic; Schizophrenia, Residual, Subchronic with AcuteExacerbation; Schizophrenia, Residual, Chronic with Acute Exacerbation;Schizophrenia, Residual, in Remission; Schizophrenia, Residual,Unspecified; Delusional (Paranoid) Disorder; Brief Reactive Psychosis;Schizophreniform Disorder; Schizoaffective Disorder; Induced PsychoticDisorder; Psychotic Disorder NOS (Atypical Psychosis); Bipolar Disorder,Mixed, with Psychotic Features; Bipolar Disorder, Manic, with PsychoticFeatures; Bipolar Disorder, Depressed, with Psychotic Features; BipolarDisorder NOS; Major Depression, Single Episode, or Recurrent withPsychotic Features; Personality Disorders, Paranoid; PersonalityDisorders, Schizoid; Personality Disorders, Schizotypal; PersonalityDisorders, Antisocial; Personality Disorders, Borderline, AnxietyDisorders, Panic Disorder, Panic Disorder with Agoraphobia, PanicDisorder without Agoraphobia, Agoraphobia without History of PanicDisorders, Social Phobia, Simple Phobia, Obsessive Compulsive Disorder,Post-Traumatic Stress Disorder, Generalized Anxiety Disorder, AnxietyDisorder NOS, Organic Anxiety Disorder, Psychoactive Substance AnxietyDisorder, Separation Anxiety Disorder, Avoidant Disorder of Childhood orAdolescence, and Overanxious Disorder.

One or more inhibitors of neurogenic NAALADase, including particularlyneurotropic β-lactam antibiotics exhibiting carboxypeptidase Einhibition activity, or β-lactamase inhibitors can be used alone, incombination or in combination with P-glycoprotein inhibitors to treatthe following psychotic conditions: Schizophrenia, Catatonic,Subchronic; Schizophrenia, Catatonic, Chronic; Schizophrenia, Catatonic,Subchronic with Acute Exacerbation; Schizophrenia, Catatonic, Chronicwith Acute Exacerbation; Schizophrenia, Catatonic, in Remission;Schizophrenia, Catatonic, Unspecified; Schizophrenia, Disorganized,Subchronic; Schizophrenia, Disorganized, Chronic; Schizophrenia,Disorganized, Subchronic with Acute Exacerbation; Schizophrenia,Disorganized, Chronic with Acute Exacerbation; Schizophrenia,Disorganized, in Remission; Schizophrenia, Disorganized, Unspecified;Schizophrenia, Paranoid, Subchronic; Schizophrenia, Paranoid, Chronic;Schizophrenia, Paranoid, Subchronic with Acute Exacerbation;Schizophrenia, Paranoid, Chronic with Acute Exacerbation; Schizophrenia,Paranoid, in Remission; Schizophrenia, Paranoid, Unspecified;Schizophrenia, Undifferentiated, Subchronic; Schizophrenia,Undifferentiated, Chronic; Schizophrenia, Undifferentiated, Subchronicwith Acute Exacerbation; Schizophrenia, Undifferentiated, Chronic withAcute Exacerbation; Schizophrenia, Undifferentiated, in Remission;Schizophrenia, Undifferentiated, Unspecified; Schizophrenia, Residual,Subchronic; Schizophrenia, Residual, Chronic; Schizophrenia, Residual,Subchronic with Acute Exacerbation; Schizophrenia, Residual, Chronicwith Acute Exacerbation; Schizophrenia, Residual, in Remission;Schizophrenia, Residual, Unspecified; Delusional (Paranoid) Disorder;Brief Reactive Psychosis; Schizophreniform Disorder; SchizoaffectiveDisorder; Induced Psychotic Disorder; Psychotic Disorder NOS (AtypicalPsychosis); Bipolar Disorder, Mixed, with Psychotic Features; BipolarDisorder, Manic, with Psychotic Features; Bipolar Disorder, Depressed,with Psychotic Features; Bipolar Disorder NOS; Personality Disorders,Paranoid; Personality Disorders, Schizoid; Personality Disorders,Schizotypal; Personality Disorders, Antisocial; Personality Disorders,Borderline.

Examples of psychotic conditions which are most preferredly treated inaccordance with the method of this invention include Schizophrenia,Catatonic, Subchronic; Schizophrenia, Catatonic, Chronic; Schizophrenia,Catatonic, Subchronic with Acute Exacerbation; Schizophrenia, Catatonic,Chronic with Acute Exacerbation; Schizophrenia, Catatonic, in Remission;Schizophrenia, Catatonic, Unspecified; Schizophrenia, Disorganized,Subchornic; Schizophrenia, Disorganized, Chronic; Schizophrenia,Disorganized, Subchronic with Acute Exacerbation; Schizophrenia,Disorganized, Chronic with Acute Exacerbation; Schizophrenia,Disorganized, in Remission; Schizophrenia, Disorganized, Unspecified;Schizophrenia, Paranoid, Subchronic; Schizophrenia, Paranoid, Chronic;Schizophrenia, Paranoid, Subchronic with Acute Exacerbation;Schizophrenia, Paranoid, Chronic with Acute Exacerbation; Schizophrenia,Paranoid, in Remission; Schizophrenia, Paranoid, Unspecified;Schizophrenia, Undifferentiated, Subchronic; Schizophrenia,Undifferentiated, Chronic; Schizophrenia, Undifferentiated, Subchronicwith Acute Exacerbation; Schizophrenia, Undifferentiated, Chronic withAcute Exacerbation; Schizophrenia, Undifferentiated, in Remission;Schizophrenia, Undifferentiated, Unspecified; Schizophrenia, Residual,Subchronic; Schizophrenia, Residual, Chronic; Schizophrenia, Residual,Subchronic with Acute Exacerbation; Schizophrenia, Residual, Chronicwith Acute Exacerbation; Schizophrenia, Residual, in Remission;Schizophrenia, Residual, Unspecified; Delusional (Paranoid) Disorder;Brief Reactive Psychosis; Schizophreniform Disorder; SchizoaffectiveDisorder; Personality Disorders, Schizoid; and Personality Disorders,Schizotypal.

In one preferred aspect of this invention there is provided a treatmentfor anxiety. Examples of anxiety disorders which are treated using thepresent method and pharmaceutical formulations of this invention,include Anxiety Disorders, Panic Disorder, Panic Disorder withAgoraphobia, Panic Disorder without Agoraphobia, Agoraphobia withoutHistory of Panic Disorders, Social Phobia, Simple Phobia, ObsessiveCompulsive Disorder, Post-Traumatic Stress Disorder, Generalized AnxietyDisorder, Anxiety Disorder NOS, Organic Anxiety Disorder, PsychoactiveSubstance Anxiety Disorder, Separation Anxiety Disorder, AvoidantDisorder of Childhood or Adolescence, and Overanxious Disorder.

Examples of the anxiety disorders which are most preferredly treatedinclude Panic Disorder; Social Phobia; Simple Phobia; Organic AnxietyDisorder; Obsessive Compulsive Disorder; Post-traumatic Stress Disorder;Generalized Anxiety Disorder; and Anxiety Disorder NOS.

The compounds used as the neurochemically functional agent in themethods and formulations of the present invention are, in one embodimentof the invention, characterized particularly by their binding topenicillin-binding proteins [as determined using methods described, forexample, by B. G. Spratt, Properties of the penicillin-binding proteinsof Escherichia coli K12, Eur. J. Biochem., 72:341-352 (1977) and N. H.Georgopapadakou, S. A. Smith, C. M. Cimarusti, and R. B. Sykes, Bindingof monolactams to penicillin-binding proteins of Escherichia coli andStaphylococcus aureus: Relation to antibacterial activity, Antimocrob.Agents Chemother., 23:98-104 (1983)] and, in the case of antibiotics, bytheir inhibition of selective carboxypeptidase and/or transpeptidaseactivity on peptide substrates comprising the amino acid sequenceAla-D-γ-Glu-Lys-D-alanyl-D-alanine. Such compounds include particularly,β-lactam compounds, including penicillins, cephalosporins, andmonocyclic and bicyclic analogs and/or derivatives thereof. Commerciallyavailable antibiotics for use in the methods and manufacture ofpharmaceutical formulations of this invention include penams, cephems,1-oxa-1-dethia cephems, clavams, clavems, azetidinones, carbapenems,carbapenems and carbacephems.

In one preferred embodiment of the present invention the peptidaseinhibitor is a compound of the formula:

wherein R is hydrogen, a salt forming group or an active ester forminggroup; R¹ is hydrogen or C₁-C₄ alkoxy; X is O, S═O, SO₂, or C; T isC₁-C₄ alkyl, halo (including chloro, fluoro, bromo and iodo), hydroxy,O(C₁-C₄)alkyl, or —CH₂B wherein B is the residue of a nucleophile B:H,and Acyl is the residue of an organic acid Acyl OH.

Examples of such commercially available compounds (1-alkoxy-1-dethiacephems) are moxalactam and flomoxef. Moxalactam is described andclaimed in U.S. Pat. No. 4,323,567. Moxalactam is particularly preferreddue to its good blood-brain barrier transport thus providing arelatively high concentration ratio of that compound in the brainrelative to blood/serum levels.

In another embodiment invention moxalactam or another commerciallyavailable β-lactam antibiotic (or derivative or analogue thereof)detailed for parenteral administration to achieve clinically effectiveantibiotic tissue concentrations, is converted to the correspondingmono- or bis-active esters to improve oral absorption of said compoundsto a level sufficient to inhibit neurogenic peptidase activity in thebrain and concomitantly effect behavior and cognitive performance,albeit at a serum concentration insufficient for clinical antibioticefficacy.

Examples of suitable in vivo hydrolysable (active) ester groups include,for example, acyloxyalkyl groups such as acetoxymethyl,pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl,1-(cyclohexylcarbonyloxy) prop-1-yl, and (1-aminoethyl)carbonyloxymethyl; alkoxycarbonyloxyalkyl groups, such asethoxycarbonyloxymethyl and alpha-ethoxycarbonyloxyethyl;dialkylaminoalkyl groups, such as ethoxycarbonyloxymethyl and(3-ethoxycarbonyloxyethyl; dialkylaminoalkyl especially di-loweralkylamino alkyl groups such as dimethylaminomethyl, dimethylaminoethyl,diethylaminomethyl or diethylaminoethyl:2-(alkoxycarbonyl)-2-alkenylgroups such as 2-(isobutoxycarbonyl) pent-2-enyl and2-(ethoxycarbonyl)but-2-enyl; lactone groups such as phthalidyl anddimethoxyphthalidyl; and esters linked to a second β-lactam antibioticor to a β-lactamase inhibitor. One example of such chemical modificationof a commercially available parenteral β-lactam antibiotic is moxalactam(Ia, Y═OH, R₁═OCH₃, and V═COM wherein, M=OH) is the preparation one ofits active ester analogue Ia wherein Y═OM, M=H or an active ester, e.g.,1-indanyl and V═CO₂M wherein M is H or an active ester and wherein atleast one of V and Y include an active ester moiety.

Suitable pharmaceutically acceptable salts of the carboxy group of theabove identified β-lactam antibiotics include metal salts, e.g.aluminum, alkali metal salts such as sodium or potassium, alkaline earthmetal salts such as calcium or magnesium, and ammonium or substitutedammonium salts, for example those with lower alkylamines such astriethylamine, hydroxy-lower alkylamines such as 2-hydroxyethylamine,bis-(2-hydroxyethyl)amine or tris-(2-hydroxyethyl)amine,cycloalkylamines such as dicyclohexylamine, or with procaine,dibenzylamine, N,N-dibenzylethylenediamine, 1-ephenamine,N-methylmorpholine, N-ethylpiperidine, N-benzyl-β-phenethylamine,dehydroabietylamine, N.N′-bisdehydro-abietylamine, ethylenediamine, orbases of to pyridine type such as pyridine, collidine or quinoline, orother amines which have been used to form salts with known penicillinsand cephalosporins. Other useful salts include the lithium salt andsilver salt. Salts within compounds of formula (I), may be prepared bysalt exchange in conventional manner.

In another embodiment of the present invention a penicillin orpenicillin analog of the formula

is employed wherein said formula X═O, S, SO, SO₂ or C; R is H or apharmaceutical acceptable salt-forming or ester-forming group; R¹ is Hor lower alkoxy, G is hydrogen or hydroxy, and Z is amino, acylamino,CO₂M, SO₃M, PO₃M₂ or PO₂M wherein M is hydrogen or a pharmaceuticallyacceptable salt-forming or ester-forming group, preferably an activeester-forming group.

Non-antibiotic or weakly antibiotic penam and cephem or cephemsulfoxides and sulfones and structurally related β-lactamase inhibitorssuch as tazobactam, clavulanic acid and sulbactam, are particularlyuseful in applications where development of antibiotic resistance is ofconcern.

Animal tests indicate a threshold effective dose of moxalactam(administered parenterally) to be about 50 μg/kg of body weight. Basedon animal test data and on the known distribution of parenterallyadministered moxalactam between the brain and other body tissues, thatthe effective minimum neurogenic peptidase inhibiting, concentration ofmoxalactam in the brain is about 30 nM. Clavulanic acid has been shownto be an effective inhibitor of neurogenic NAALADase when administeredi.p. at less than 1 microgram per kilogram of body weight. The range ofeffective dosage levels of the inhibitors when used in the treatment ofbehavioral and/or cognitive disorders in accordance with this inventionwill depend significantly on patient body weight, the affinity of theinhibitor for the target neurogenic protease, the blood-brain barriertransport characteristics of the active compound, the mode ofadministration and the optional use of available drugformulations/conjugation technologies available for enhancement ofblood-brain barrier transport. For parenterally administered moxalactamthe minimum effective dose in hamsters and other test species is about50 micrograms per kg of body weight, more or less. The use of moxalactamin an oral dosage form, preferably modified or derivatized in the formof an active ester, is estimated to range from about 2.5 to about 50 mgper dose, much less than the dose of moxalactam necessary to providetherapeutically effective antibiotic concentration. The effective oraldose of clavulanate is expected to be about 0.1 to about 10 mgs perdose. Clavulanate is orally absorbed and it exhibits good blood brainbarrier transport.

The effective doses of other peptidase inhibitors will vary, againdepending on their inherent affinity for the target peptidase, theselected route of administration, patient weight, and blood-brainbarrier transport efficiency. The effective dosages of peptidaseinhibitors used in accordance with the present invention can be readilydetermined empirically using animal models coupled with use of artrecognized analytical techniques. Typically, the dosage levels forβ-lactam antibiotic compounds used in the methods and formulations ofthis invention is less than that necessary to achieve clinicallyeffective antibacterial levels. Parenteral dosages of β-lactamantibiotic compounds can range from about 1 to about 80 mg per dose.Oral dosages can range from about 2.5 to about 150 mg per dose. Higheror lower dosage amounts may be appropriate and used in accordance withthis invention when patient circumstances dictate such in the judgmentof the attending physician. Thus, for example, where patient/clinicalconditions are such that the inherent antibiotic activity of theβ-lactam compounds are not considered to be a complicatingcontraindication, higher doses of the antibiotic up to or exceeding thedosage levels capable of providing threshold clinically effectiveantibiotic blood levels can be used to treat patients in need of therapyeffected by peptidase inhibition in accordance with this invention.

The present invention further provides certain pharmaceuticalformulations for treatment of behavioral or cognitive disorders andother disease states associated with production of abnormal glutamateconcentrations in nervous tissues and other tissues harboring NAALADaseactivity. Generally the formulation comprises a neurologically activeingredient including a compound capable of inhibiting a bacterial enzymeand capable of inhibiting a neurogenic peptidase that is known, byempirical evidence, to selectively act on a peptide comprising the aminoacid sequence Ala-D-γ-Glu-Lys-D-alanyl-D-alanine, and a pharmaceuticallyacceptable carrier therefor. In one embodiment the pharmaceuticalformulation in a unit dosage form comprises an amount of a β-lactamcompound capable of inhibiting peptidase activity in a patientexperiencing or disposed to develop a neurological condition that couldbe prevented or treated to reduce its symptoms by peptidase inhibition.The amount of the peptidase inhibitor and the nature of the carrier isdependent, of course, on the intended route of administration. Theamount of inhibitor is that amount effective to provide upon delivery bythe predetermined route of administration, a concentration of theinhibitor in the tissue where peptidase inhibition is desired, e.g., inthe brain effective to treat and reduce symptoms of the targetedbehavioral or cognitive disorders or other disorders than can be treatedby inhibition of peptidase activity. In embodiments utilizing β-lactamantibiotic compounds the amount of the peptidase inhibitor in thepresent formulations is typically less than that capable of providingclinically effective bacterial protease inhibition, i.e., less than thatcapable of providing antibiotically effective levels when administeredto a patient in the dosage form provided. The peptidase inhibitors foruse in accordance with this invention can be combined with one or morepharmaceutically acceptable carriers, and may be administered, forexample, orally in such forms as tablets, capsules, caplets, dispersiblepowders, granules, lozenges, mucosal patches, sachets, and the like. Theinhibitor can be combined with a pharmaceutically acceptable carrier,for example starch, lactose or trehalose, alone or in combination withone or more tableting excipients and pressed into tablets or lozenges.Optionally, such tablets, caplets or capsules can be enterically coatedto minimize hydrolysis/degradation in the stomach. Oral dosageformulations contain about 1 to about 99% by weight active ingredientand about 1 to about 99% of a pharmaceutically acceptable carrier and/orformulating excipients. Optionally, when β-lactam antibiotics are usedas the inhibitors the dosage forms can be formulated by combining itwith a P-glycoprotein inhibitor to provide enhanced drug half-life andbrain concentrations of the active ingredient. Alternatively, theprotease inhibitor can simply be co-administered with a P-glycoproteinor β-lactamase inhibitor.

In another embodiment of the invention pharmaceutical preparations maycontain, for example, from about 2.5% to about 90% of the activeingredient in combination with the carrier, more usually between about5% and about 60% by weight active ingredient. The pharmaceuticalformulations in accordance with one embodiment of this invention areformulated for per os administration, i.e., oral ingestionadministration or buccal or sublingual administration (in the form ofsachets, lozenges, and/or oral mucosal patches). In another embodimentthe dosage form is formulated for per os administration in a prolongedrelease dosage form formulated to release the active ingredient over apredetermined period of time.

Topical dosage forms, including transdermal patches, intranasal, andsuppository dosage unit formulations containing the active peptidaseinhibitor and conventional non-toxic pharmaceutically acceptablecarriers, adjuvants and vehicles adapted for such routes ofadministration are also within the scope of this invention.

The pharmaceutical formulations in accordance with this inventionalternatively can be delivered via parenteral routes of administration,including subcutaneous administration, intraperitoneal administration,intramuscular administration and intravenous administration. Suchparenteral dosage forms are typically in the form of aqueous solutionsor dispersions utilizing a pharmaceutically acceptable carrier such asisotonic saline, 5% glucose, or other well known pharmaceuticallyacceptable liquid carrier composition.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders or lyophilizatesfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the dosage form must be sterile and it mustbe stable under the conditions of manufacture and storage, and must bepreserved against the contaminating action of microorganisms. Thecarrier for injectable formulations can be a solvent or dispersionmedium containing, for example, water, ethanol, or a polyol (or exampleglycerol, propylene glycol and liquid polyethyleneglycol), mixturesthereof, and vegetable oil.

Parenteral dosage forms of the peptidase inhibitors useful for treatmentof behavioral and cognitive disorders and other disease statesresponsive to neurogenic peptidase inhibition can also be formulated asinjectable prolonged release formulations in which the proteaseinhibitor is combined with one or more natural or syntheticbiodegradable or biodespersible polymers such as carbohydrates,including starches, gums and etherified or esterified cellulosicderivatives, polyethers, polyesters (particularly polylactide,polygylcolide or poly-lactide-glycolides), polyvinyl alcohols, gelatins,or alginates. Such dosage formulations can be prepared, for example, inthe form of microsphere suspensions, gels (of hydrophilic or hydrophobicconstitution), or shaped-polymer matrix implants that are well-known inthe art for their function as “depot-type” drug delivery systems thatprovide prolonged release of the biologically active components. Suchcompositions can be prepared using art-recognized formulation techniquesand designed for any of a wide variety of drug release profiles.

The administration of pharmaceutical compositions for use in the presentinvention can be intermittent or at a gradual, or continuous, constantor controlled rate to a patient in need of treatment. In addition, thetime of day and the number of times of day that the pharmaceuticalformulation is administered can vary depending on the patient conditionand environment. The level of efficacy and optimal dosage and dosageform for any given peptidase inhibitor for use within the scope of thisinvention is patient-dependent and adjustable within reasonable rangesin the judgment of the attending physician. The formulation is typicallyadministered over a period of time sufficient to treat or prevent thepatient disease state, e.g., to modify the behavioral or cognitiveperformance of the patient undergoing treatment. The peptidase inhibitorformulations may be continued to be administered using the same orattenuated dosage protocol for prophylaxis of the targeted diseasestate.

The above-described embodiments of the present invention derive in partfrom the mechanism of action deduced from data gathered in animalbehavioral cognitive and skill models described below. Other embodimentsof the invention will be apparent from analysis of the data obtained inthe following non-limiting experimental examples, which are butillustrative of the behavior modification and cognitive performance andimprovement attainable by use of the method and formulations of thepresent invention.

Experimental Examples

Marketed in 1981-1982 moxalactam (Mox) was employed widely in the worldas a third-generation cephalosporin-like antibiotic. Clinical efficacyand safety were evaluated in over 2200 patents with bacterial infections(Jackson et al. 1986). Of the 260 patents treated with Mox forgram-negative meningitis, 241 (93%) showed satisfactory response toantibiotic therapy. Patents were treated with 4 g of Mox every 8 hrs for2-3 weeks. Peak plasma concentrations occur within an one hr after IMinjection with an elimination half-life of 2.3 hrs. There is noaccumulation with multiple injections occurring at 8-12 hr intervals.Moxalactam can penetrate the blood brain barrier. Cerebrospinal fluid(CSF) levels of Mox range from 25-39 μg/ml following a 2.0 g IV dose ofdrug. The CSF concentration as a percentage of serum concentration isestimated to be 20%. The D isomer has antibacterial activity and has agreater unbound fraction to plasma protein that the L isomer.

Behavioral Studies with Moxalactam Methods Animal Care

Male Syrian golden hamsters (Mesocricetus auratus) (140-150 g) obtainedfrom Harlan Sprague-Dawley Laboratories (Indianapolis, Ind.) were housedindividually in Plexiglas cages (24 cm×24 cm×20 cm), maintained on areverse light:dark cycle (14L:10D; lights on at 19:00 hr) and providedfood and water ad libitum. Animals were acclimated to the reverselight:dark cycle for at least two weeks before testing. All behavioraltests were conducted during the dark phase of the circadian cycle. Allanimals were acquired and cared for in accordance with the guidelinespublished in the Guide for the Care and Use of Laboratory Animals(National Institutes of Health Publications No. 85-23, Revised 1985).

Offensive Aggression

Agonistic behavior can be classified as either offensive or defensiveaggression (Blanchard and Blanchard, 1977; Adams, 19798; Albert andWalsh, 1984). Offensive aggression is characterized by the aggressorinitiating an attack on an opponent, while defensive aggression lacksactive approach. Both types of aggression have their own uniqueneurobehavioral systems. The stimuli that elicit offensive and defenseattack are different, as are the sequences of behaviors that accompanyeach agonistic response. While much of the empirical data supporting thenotion of unique offensive and defensive neural networks have beencollected from animal models, there are interesting and compellingsimilarities in human aggression that suggest a similar neuralorganization (Blanchard, 1984). Offensive aggression is easily studiedusing male golden hamsters tested in a resident/intruder paradigm, anestablished model of offensive aggression (Ferris and Potegal 1988).Placing an unfamiliar male hamster into the home cage of another malehamster elicits a well-defined sequence of agonistic behaviors from theresident that includes offensive aggression.

Behavioral Measures and Analysis

Hamsters are nocturnal and as such all behavioral tests were performedduring the first four hrs of the dark phase under dim red illumination.The resident was scored for offensive aggression, e.g., latency to bitethe intruder, the total number of bites, total contact time with theintruder and flank marking over a 10 min test period (Ferris andPotegal, 1988). Flank marking is a form of olfactory communication inwhich a hamsters arches its back and rubs pheromone producing flankglands against objects in the environment (Johnson, 1986). Flank markingfrequency is greatly enhanced during aggressive encounters and isparticularly robust in dominant animals initiating and winning fights(Ferris et al., 1987).

Parametric data, i.e., latencies and contact time, were analyzed with aone-way ANOVA followed by Newman-Keuls post hoc tests. Non parametricdata, i.e., number of bites and flank marks, were analyzed withKruskal-Wallis tests followed by Mann-Whitney U tests to determinedifferences between groups. Two sample comparisons were analyzed withpaired and unpaired t-Tests for parametric data and Wilcoxon andMann-Whitney Tests for paired and unpaired non-parametric data,respectively.

Results I. High Dose Moxalactam

In a pilot study, Mox (50 mg/kg in a volume of ca. 150 μl) was givenintraperitoneally (IP) to six male hamsters prescreened for aggressivebehavior toward smaller intruders. Treatments with Mox and salinevehicle were counter balanced so each animal received both treatmentsseparated by at least 48 hr. Animals were tested 90 min after treatmenta period estimated to reflect peak plasma levels of Mox (Jackson et al.1986). Moxalactam was dissolved in 0.9% NaCl and stored on ice. It wasprepared fresh for each study.

Resident animals treated with saline vehicle bite intruders in less thanone min (FIG. 1). Following Mox treatment the mean latency to bite wasincreased to over six min (p<0.05). In addition, the number of bitesover the 10 min observation period were significantly reduced (p<0.05).However, the contact time, i.e., the time the resident spent smellingand exploring the intruder was also significantly reduced (p<0.01). Thedecrease in flank marking did not reach significance but there was atrend (p<0.07).

SUMMARY

The general decrease in all behavioral measures associated withoffensive aggression raises the possibility that the 50 mg/kg dose ofMox has non specific depressive effects on motor activity and arousal.To examine this possibility, it was necessary to run dose responsestudies to find the lowest dose of Mox that effectively inhibitsoffensive aggression without altering other behaviors.

II. Moxalactam Dose Response

To find the lowest dose of Mox that could significantly reduce offensiveaggression, a range of concentrations (vehicle, 0.5, 5.0, 50, 500, and5000 μg/kg) were tested in six animals (FIGS. 2 & 3) The treatments werecounter balanced with each animal receiving each treatment separated byat least 48 hrs. The latency to bite was significantly different betweentreatments (F (5,30)=5.66; p<0.001). Moxalactam treatment with doses of5.0 μg and less had no effect on any behavioral measures of offensiveaggression. However, the dose of 50 μg/kg significantly delayed bitelatency by over seven min (p<0.001) as compared to vehicle control.Doses of 500 μg and 5.0 mg also significantly increased bite latency. Aswas expected, the same doses that increased bite latency also decreasedthe number of bites (H=24.12; p<0.001). Animals treated with 50 μg Moxshowed a significant reduction in bites (p<0.05). Indeed, three of sixanimals never bite at all in the 10 min observation period. The contacttime was significantly different between treatments (F (5,30)=2.5;p<0.05). Doses of 500 μg and 5 mg significantly reduced contact time ascompared to vehicle control (p<0.05 and p<0.01, respectively). Flankmarking was not significantly different between groups (H=9.256;p<0.09).

SUMMARY

These data identify the dose of 50 μg/kg of Mox as very effective ininhibiting offensive aggression without significantly reducing contacttime and flank marking. Higher doses of Mox, while effective in reducingmeasures of aggression also reduced contact time. Hence, the 50 μg dosewould appear to be best for future behavioral tests. Having identifiedthe most effective dose of Mox a more thorough study using a greaternumber of animals and a greater spectrum of behavioral tests wasnecessary.

III. Behavioral Tests with 50 Mg Moxalactam

Offensive Aggression

Thirteen hamsters were tested for offensive aggression followingtreatment with saline vehicle or 50 μg/kg Mox (FIG. 4). Both treatmentswere given IP in a volume of ca. 150 μl. Animals were tested 90 minafter injection. Each animal received both treatments. The order ofinjections was counter balanced with no less than 48 hrs betweentreatments. Moxalactam significantly increased bite latency (p<0.001)and reduced the number of bites (p<0.01). There was no significantchange in contact time or flank marking.

SUMMARY

This larger study of low dose Mox corroborates the dose-response studyconfirming that Mox can effectively reduce offensive aggression withoutaltering social behavior as measured by the time spent with theintruder.

Motor Activity in an Open Field

Six animals were tested for general motor activity in an “open field”following treatment with saline vehicle or 50 μg/kg Mox (FIG. 5). Thestudy was counter balanced with each animal receiving each treatment.Ninety minues after injection a single animal was placed into a largeclean Plexiglas cage (48×32×40 cm) devoid of any bedding. This openfield was delineated into equal quadrants by tape on the underside ofthe cage. Animals were scored for motor activity by counting the numberof quadrants traversed in 1 min. There was no significant differencebetween treatments on open field activity.

Olfactory Discrimination

Sixteen animals were treated with vehicle or 50 μg/kg Mox and tested forolfactory discrimination by measuring their latency to find hiddensunflower seeds (FIG. 5). The injections were counterbalanced with eachanimal receiving each treatment. Prior to testing animals were fastedfor 24 hrs. Ninety minutes after injection animals were briefly takenfrom their home cage while six sunflower seeds were buried under thebedding in one corner. Animals were placed back into their home cage andscored for the latency to find the seeds in a ten min observationperiod. The latency to find the seed was significantly (p<0.001) reducedin animals treated with Mox as compared to vehicle controls.Surprisingly, all seeds were rapidly consumed in less than five minfollowing treatment with Mox but not saline. In fact, not one of thesixteen animals consumed all of the seeds following saline as comparedto all animals treated with Mox.

Sexual Activity

Six animals were tested over a five min observation period for sexualactivity following treatment with saline vehicle or 50 μg/kg Mox (FIG.6). The study was counter balanced with each animal receiving eachtreatment. Ninety min after injection, animals were scored for latencyto mount and number of intromissions, i.e., bouts of copulation, towarda receptive female placed into their home cage. Female golden hamsterswere ovariectomized under general anesthesia. Following recovery animalswere treated with a single SC injection of 50 mg estradiol benzoate forthree consecutive days to induce sexual receptivity. On the day oftesting the estrogen primed females were introduced into the home cageof the experimental males. The first investigation by the malesroutinely caused robust lordosis in the female. Lordosis, is astereotyped posture characterized by intense, sustained vertebraldorsiflexion.

Following vehicle treatment, animals mounted and thrust a receptivefemale in ca. 30 sec. The time to mount was significantly increased(p<0.05) following treatment with Mox. While both treatments showed highbout of copulation, animals treated with Mox showed a trend toward adecreased intromission rate (p<0.07).

SUMMARY

Moxalactam appears to have a very good serene profile. Serenics aredrugs used to treat impulsivity and violence (Olivier and Mos, 1991).Serenics should suppress offensive aggression without interfering withsocial, appetitive and cognitive behaviors. Social interest in anintruder, i.e. contact time is not altered by Mox. Flank marking andactivity in an open field is also unaltered with drug treatment evidencethat general arousal and motor activity is normal. Fasted animalstreated with Mox are better able to find hidden sunflower seeds evidencethat drug treatment does not interfere with olfaction or motivation tofind food; in fact, it may enhance it. Interestingly, Mox treatmentreduced the latency to mount a receptive female and lessened, althoughnot significantly, the bouts of copulation in a five min observationperiod. It should be noted that Mox treated animals were still verysexually active, except the behavior appeared less intense. Thisantiaggressive effect of Mox combined with a mollification of sexualactivity might have therapeutic value in treating violent sex offenders.

Development of eltoprazine, one of the first serenics, was abandoned, inpart, because it was found to increase fear and anxiety in animals(Olivier et al 1994). To control for this possibility, it was necessaryto test Mox in a model used to screen drugs for their affect on anxiety

IV. Testing Moxalactam for Anxiolytic Activity Elevated Plus-Maze

The elevated plus-maze was developed for the detection of anxiolytic andanxiogenic drug effects in the rat (Pellow et al., 1985). The method hasbeen validated behaviorally, physiologically, and pharmacologically. Theplus-maze consists of two open arms and two enclosed arms. Rats willnaturally make fewer entries into the open arms than into the closedarms and will spend significantly less time in open arms. Confinement tothe open arms is associated with significantly more anxiety-relatedbehavior and higher stress hormone levels than confinement to the closedarms. Clinically effective anxiolytics e.g., chlordiazepoxide ordiazepam, significantly increase the percentage of time spent in theopen arms and the number of entries into the open arms. Conversely,anxiogenic compounds like yohimbin or amphetamines reduce open armentries and time spent in the open arms.

Method

Male Wistar rats weighing 250-300 g were group housed in a normal 12:12light-dark cycle light on at 0800 hr and provide food and water adlibitum. The plus-maze consisted of two open arms, 50×10 cm, and twoenclosed arms 50×10×40 cm with an open roof, arranged such that the twoopen arms were opposite to each other. The maze was elevated to a heightof 50 cm.

Eight animals were tested in the plus-maze 90 min following IP injectionwith 50 μg/kg Mox and saline vehicle. The order of treatments wascounter balanced with at least 48 hrs between injections. At the startof the experiment the animal was place in the center of the plus mazefacing the closed arm. Over a five min observation period, animals werescored for the latency to enter the closed arm, time spent in the closedarm and the number of open arm entries following the first occupation ofthe closed arm (FIG. 7). Treatment with Mox significantly increased thelatency to enter the closed arm (p<0.05) as compared to vehicle. Thetime spent in the closed arm was significantly reduced (p<0.01), whilethe number of open arm entries was significantly elevated (p<0.05).

SUMMARY

These data show Mox given at a dose of 50 μg/kg has anxiolytic activity.This finding enhances the serenic profile of Mox and delineates it fromprevious serenics like eltoprazine that suppressed offensive aggression,in part, by increasing fear and anxiety. These data also show that Moxmay have therapeutic value as an anxiolytic

However, the anxiolytic activity of Mox raises other concerns aboutbehavioral specificity. Many anxiolytics, particularly thebenzodiazepines are sedatives and can depress general motor activity andmay also acts as amnesics and interfere with learning and memory. SinceMox was show to have no effect of flank marking or activity in an openfield it is unlikely to act as a general sedative. However, it wasnecessary to test Mox for any untoward effects on learning and memory.

V. Testing Moxalactam For Anxiolytic Activity Moxalactam V.Chlordiazepoxide Methods

Because Mox and CDP have different bioavailability profiles, e.g. brainpenetrance, their CNS activity could not be compared by giving systemicinjections of equimolar concentrations of each drug. Instead it wasnecessary to give both drugs directly into the cerebroventricular systemto by pass the blood brain barrier. Animals were anesthetized withsodium pentobarbital (50 mg/kg), implanted with microinjection guidecannulae aimed at the lateral ventricle and allowed to recover for twodays before testing. To groups of six animals each were tested with Moxor CDP. Each animals received a injection of drug and 0.9% NaCl vehicleon two separate days. The order of injections was counterbalanced andseparated by two days. Both Mox and CDP were prepared in 0.9% NaCl at aconcentration of 1 mM. All injections were given in a volume of 2 ulover 10 secs in fully conscious, restrained animals. Sixty min lateranimals were tested in the plus-maze for a 3 min observation period andscored for behaviors as noted previously.

Results

Mox treatment significantly (p<0.05) delayed the time it took to enterthe closed arm as compared to vehicle treatment (FIG. 17). Treatmentwith Mox caused animals to spend most of their time in the light arms ofthe plus-maze. Time spent in the dark was significantly (p<0.01) lowerfollowing Mox treatment as compared to vehicle. Treatment of CDP at the1 mM concentration had no effect on either the latency to enter theclosed arm or time spent in the closed arm as compared to vehicletreatment.

Controlling for Non-Specific Depression of Motor Activity

When CDP is given systemically to rodents in doses of 5-15 mg/kg it is asedative and depresses motor activity. However, this depression of motoractivity disappears following repeated administration of CDP overseveral days. Only after the animals become insensitive to the motoreffects of CDP are they tested in the plus maze for anxiolytic activity.To control for any non-specific effects of Mox and CDP on motor activityfollowing their direct injection into the brain, animals were tested inthe open field 30 min prior to testing in the plus (FIG. 18). There wasno significant effect for either anxiolytic on general motor activity.

SUMMARY

The finding that Mox is an anxiolytic enhances its serenic profile anddelineates it from previous serenics like eltoprazine that suppressedoffensive aggression, in part, by increasing fear and anxiety. On anequimolar basis, Mox showed anxiolytic activity given directly into thebrain as compared to CDP which had none. These data show that Mox mayhave therapeutic value as an anxiolytic in addition to a serenic.

However, the anxiolytic activity of Mox raises other concerns aboutbehavioral specificity. Many anxiolytics, particularly thebenzodiazepines are sedatives and can depress general motor activity andmay also acts as amnesics and interfere with learning and memory. SinceMox was show to have no effect of flank marking or activity in an openfield it is unlikely to act as a general sedative. However, it wasnecessary to test Mox for any untoward effects on learning and memory.

VI. Testing Moxalactam for Spatial Memory Radial Arm Maze

The radial arm maze is one of the most commonly used methods for testingspatial learning and memory in rodents. Developed by Olton andco-workers (1976), it provides the simultaneous choice of severalalternative paths for the test subject. Animals must learn whichlocations provide food (place learning) using visuospatial cues.

Methods

Experimental Trials: The experimental trials consist of three phases(described below). The arms of the maze are numbered clock-wise from oneto seven with arm number one being the arm furthest to the right side ofthe maze. All trials are ca. 12 min long. When not being tested, allhamsters have unlimited access to water. In addition to the sunflowerseeds in the maze, hamsters are given one Agway Prolab 3000 food pelletdaily. Trials within all the phases are conducted on successive days.

Phase One: Phase One consists of five 15 min trials. Prior to thebeginning of each of the five trials in Phase One, four sunflower seedsare placed at the ends of arms one, two, and three. Arms four, five,six, and seven remain empty.

Phase Two: Phase Two of the experimental trials are identical to PhaseOne except that the seeds are placed in arms two, four, and seven. Armsone, three, five and six remain empty. Phase Two consists of four 15 mintrials.

Phase Three Phase Three of the experimental trials consists of three 15min trials, with arm two, four, and seven baited with sunflower seeds.Phase Three differ form Phase Two in that the maze is rotated clockwisein the room 110°.

Coding of Behaviors: An arm entry was scored if all four paws of ahamster crossed an arm threshold. A full arm entry into an awl is scoredif a hamster's snout touches the top of the block at the end of an armor if their snout passes the block. These scores were made for baitedand unbaited arms. In addition, the number of seeds pouched by thehamsters was scored.

Results

Six male hamsters were tested in the radial arm maze following treatmentwith 0.9% NaCl or 50 μg/kg Mox (FIG. 8). Each animal received eachtreatment and the order of treatments was counter balanced. The mostcritical measure in the radial aim maze is the number of seedsdiscovered after reversing the orientation of the maze on the final dayof testing. Moxalactam treatment significantly increased seed finding(p<0.01) as compared to vehicle treatment.

SUMMARY

These data support the notion that the anxiolytic profile of moxalactamis not accompanied by any disruption in learning and memory as is thecase with benzodiazepine anxiolytics. On the contrary, moxalactamenhances spatial memory would may act as a psychotropic agent to improvecognitive performance. This finding suggests that moxalactam may be aneffective therapeutic agent for the treatment of ADHD and conductdisorder in children and senility in geriatric patients.

Spatial Navigation in Water Maze

The Morris water maze like the radial arm maze was developed to testspatial memory (Morris, 1984). The pool is divided into quadrantsusually designated North, South, East and West. The water in the pool ismade opaque with milk powder. Hidden just beneath the surface in one ofthe quadrants is a platform that serves as a escape route for rodentsplaced into the pool. An animal is placed some where in the pool from avariety of different start points and is timed for latency to find theplatform, percent time spent in each quadrant, distance traveled andswimming speed. The animals has no visual or spatial cues in the pooland must rely on extra-maze cues, i.e., objects set up outside the poolthat can be seen by the swimming animal. Through a series of trials arat develops “place learning” or knowledge about the position of theplatform based upon the extra-maze cues. The platform can be moved to adifferent quadrant each day combining spatial memory with workingmemory. This paradigm involves extinction of the prior memory andresolution of a new spatial problem.

Methods

The water maze consisted of a black plastic circular pool ca. 150 cm indiameter and 54 cm in height filled to a level of 35 cm with water madeopaque with powdered milk. The pool was divided into four quadrants witha platform 10 cm in diameter submerged 2 cm below the surface in thenorthwest quadrant. The water was maintained at a temperature of 25° C.Around the pool were several visual cues. Above the pool was a videocamera for tracking the movement of the experimental animal. The datacollection was completely automated using the software developed by HVSImage (Hampton, UK). Before testing, rats were familiarized with thepool and platform placed in the northwest quadrant. Each day for 4consecutive days, animals were placed into pool at random sites andgiven two min to find the platform. Animals were treated one hr beforetesting with 50 μg/kg Mox (n=11) or vehicle (n=10). Following thesefamiliarization trials, animals were tested for spatial navigation. Thefirst day of testing began with the platform in the expected northwestquadrant. All behavior was videotaped for a two min observation period.After testing the animal were dried off and placed back into their homecage. On each subsequent day the platform was moved to a new quadrantand the rat started at different positions. The rat was always placedinto the pool facing the side wall. The start positions relative to theplatform were different for each of the four trials; however, theplatform was always in the same relative position in each quadrant.Twenty cm in from the side of the pool and in the left corner from thecenter facing out.

Results

A two-way ANOVA showed a significant main effect for treatment(F_((1,20))=6.48, p<0.05) and days of testing (F_((3,63))=5.76, p<0.01)(FIG. 19). There was also a significant interaction between treatmentsand testing days (F_((3,63))=4.35, p<0.01). Newman-Keuls post hoc testsshowed a significant difference between treatments on day two (p<0.05),day three (p<0.01) and day four (p<0.05) (FIG. 19). On each of thesedays Mox treated animals showed significantly shorter latencies to findthe hidden platform than the vehicle treated group. Indeed, vehicletreated animals showed a significant increase in latency on days 2(p<0.05) and 3 (p<0.01) as compared to day 1.

The strategy for finding the platform was strikingly similar for bothtreatments (FIG. 19, lower two graphs) as judged by the percentage oftime the animals spent in each quadrant. For any quadrant on any daythere was no significant difference between treatments. There was asignificant difference between days for percentage of time spent in anyparticular quadrant (e.g., North, F_((3,63))=28.80, p<0.0001). Animalsspent a significant portion of their time in certain quadrants oncertain days. For example, on Day 1 both Mox and Vehicle animals spentmost of their time in the North quadrant as compared to the otherquadrants (p<0.01). This was to be expected since they had knowledge ofthe location of the platform in this quadrant from the familiarizationprocedure. Interestingly, Vehicle animals also showed a significant(p<0.05) amount of time in the West quadrant on Day 1 as compared toSouth and East. This was probably because the platform was hidden in thenorthwest part of the North quadrant. On Day 2, Mox and Vehicle animalsspent a significant amount of time in both the North and South quadrantsas compared to East and West. On Day 3 Mox animals show no particularbias for any quadrant while Vehicle animals still show a significantinterest in the North quadrant as compared to South and West. By Day 4both Mox and Vehicle spent most of their time in the correct quadrant(West) with the least amount of time in the East quadrant where theplatform was hidden the day before. This strategy on Day 4 shows goodspatial, working and procedural memory for both treatments.

The distance covered to reach the platform across days was notsignificantly different between Mox and Vehicle animals (FIG. 20).However, Mox animals showed significantly greater swim speed thanVehicle animals (F_((1,20))=22.94, p<0.0001)(FIG. 20). For example, onDay 2 both groups traveled a similar distance to the platform except Moxanimals covered the distance at almost twice the speed (p<0.01). Whilethere was no main effect across days (F_((3,63))=2.27, p<0.09) there wasan interaction between swim speed and days (F_((3,63))=2.75, p<0.05) forMox treatment as this group decreased their swim speed over time.

Cue Navigation in Water Maze Method

On the day following the last day (Day 4) of spatial navigation, animalswere tested for cue navigation. In these tests, the platform was raisedabove water level. One hr before testing animals were treated with Moxor saline vehicle. The same animals that were treated with Mox duringspatial navigation were treated with Mox for cue navigation. Animalswere run through a series of two minute trials with 45 min betweentrials. At each trial, the platform was moved to a different quadrant.The cue navigation study was identical to the spatial navigation exceptthe platform was visible and the testing was done over five consecutivetrials done on a single day. Animals were scored for latency to find theplatform, percent time spent in each quadrant, path distance and swimspeed for all testing periods

Results

The latency to find the platform was different between Mox and Vehicletreated animals (F_((1,20))=24.68, p<0.0001) (FIG. 21). There was also amain effect for days (F_((4,84))=6.53, p<0.0001) but no interactionbetween treatment and days (F_((4,84))=0.99, p<0.4). On trials 1,3, and4 Mox animals showed significantly shorter latencies than Vehicleanimals.

As in spatial navigation, the strategy for finding the platform was verysimilar for both treatments (FIG. 21, lower two graphs) as judged by thepercentage of time the animals spent in each quadrant. For any quadranton any trial there was no significant difference between treatments(e.g., South, F_((1,20))=1.61, p<0.21). There was a significantdifference between trials for percentage of time spent in any particularquadrant (e.g., South, F_((4,84))=16.70, p<0.0001). Animals spent asignificant portion of their time in certain quadrants on certaintrials. For example, on Trial 5 both Mox and Vehicle animals spent asignificant amount of time in the North quadrant were the platform washidden, and the West quadrant were the platform had been on the previoustrial.

Unlike spatial navigation, the distance traveled during cue navigationwas significantly different between Mox and Vehicle animals(F_((1,20))=44.11 p<0.0001) (FIG. 22). There was also a significant maineffect for trials (F_((4,84))=7.90, p<0.0001) and interaction betweentreatment and trails (F_((4,84))=2.67, p<0.05). On Trial 1 there was nodifference in path length between treatments. However, on Trials 3 and 4Vehicle animals traveled significantly farther to find the platform thanMox animals. The path length did not significantly change across trialsfor Mox animals. Whereas, the mean path length on Trial 3 for Vehicleanimals was significantly greater than any other trail for thistreatment.

Unlike spatial navigation, there was no significant difference in swimspeed between the two treatments (F_((1,20))=0.67, p<0.42) (FIG. 22).However, there is a main effect across trials (F_((4,84))=17.18,p<0.0001) and an interaction between treatment and trials(F_((4,84))=4.10, p<0.01). In both treatments there is a significantincrease in swim speed over each subsequent trail. For example, fromTrial 1 to Trial 4 Mox and Vehicle animals showed a significant increasein swim speed (p<0.01).

SUMMARY

Moxalactam treated animals are more effective in finding the hidden andvisible platform in the water maze than vehicle treated controls.However, the strategy for success in each navigation paradigm wasstrikingly different. During spatial navigation, animals must rely onextramaze cues and procedural memory to find the moving platform. Moxand vehicle animals appeared to show the same learning and memory asthere was no difference in the percentage of time spent in each quadrantfor each day of testing. There was no ostensible difference in the swimpatterns (FIGS. 23 and 24). The distance traveled between treatments wasnot significantly different. Mox animals found the platform sooner, inpart, because they swam faster. However, cue navigation presented adifferent profile. Again Mox treated animals out performed vehicleanimals on latency to find the platform. Again the search strategy asdefined by the percentage of time spent in each quadrant was strikinglysimilar. However, unlike spatial navigation, animals treated with Moxshowed a much shorter path length. Moreover, both treatment groups swamat the same speed.

These data support the notion that the anxiolytic profile of moxalactamis not accompanied by any disruption in learning and memory as is thecase with benzodiazepine anxiolytics. On the contrary, moxalactamenhances spatial memory and may act as a psychotropic agent to improvecognitive performance. This finding suggests that moxalactam may be aneffective therapeutic agent for the treatment of ADHD and conductdisorder in children and senility in geriatric patients.

VII. Social Behavior in Non-Human Primates Experimental Procedure

Eight, two year old adolescent male rhesus macaques were tested withMox. Animals were raised with their mothers in a group setting at afield station. At one year of age, they were transferred to individualcages. Each day thereafter, they were paired housed for two-three hrs.The adolescent partners were always the same. This year long procedureresulted in adolescent partners or “play-mates” having a well-definedhistory of social interaction with recognizable dominant and subordinatestatus. The display of social behaviors in this arrangement are veryrobust because of the limited amount of time the monkeys spend together.

During the experiment the monkeys were paired in the “play-cage” wherethey were video taped for one hour. The study was designed so thatbehavioral data were obtained for each monkey under Mox and vehicletreatment. The treatment was an ABA type schedule of administration: Day1—one member of each pair received 0.9% NaCl vehicle, Day 2—drug, Day3—vehicle. Only one member of a pair was injected on a test day. Theother member of a pair was injected a week later according to the sameABA schedule. Moxalactam was injected LM in a dose of 1 mg/kg. Animalswere video taped sixty minutes after injection for a one hr observationperiod. Animals were scored for over forty different behaviors (Winslowet al., 1988). Only twenty-eight are listed on TABLE I. The unreportedbehaviors, e.g., self-bites, vocalizations, clinging, mounts, escapes,self grooming were so infrequent that they were omitted from theanalysis. Paired t-test was run for each behavioral measure.

Results

The duration of play fighting was significantly reduced (p<0.05) by Moxtreatment as compared to vehicle. This finding was not affected by thesocial status of the animal, i.e. both dominant and subordinate animalsshowed diminished play fighting following treatment with Mox.Interestingly, several different measures of agonistic behavior, e.g.,composite aggression scores, clustered together at near significantlevels. It should be noted that these are juvenile rhesus monkeys, andas such their expression of social aggression is primarily confined toplay fighting. The aggression does not have the same emotional valenceas adults. Nonetheless play fighting is thought to be the juvenileantecedent to adult aggression. Allogrooming for adolescent and adultmonkeys is the primary measure of affiliative behavior. While Moxsignificantly reduced the duration of play fighting it had no effect onallogrooming.

SUMMARY

Moxalactam given in a dose of 1 mg/kg to adolescent rhesus monkeyssignificantly reduces play fighting a measure of agonistic behavior.However, allogrooming the key measure of affiliative behavior isunaltered. Hence the finding that Mox can reduce agonistic behavior inrodents translates to non-human primates.

VIII. Testing D and L Isomers of Moxalactam Rationale

The 3D structure of drugs can naturally occur as mirror images orisomers. These isomers are classified as D or L based on their rotationof light. Only one of the isomers usually has biological activity. Sincethe preparation of Mox used in these studies is a mixture of the twoisomers it was necessary to isolated and test for the active isomer.

Methods

Moxalactam sodium salt (FW 564.4) was obtained as a mixed isomer fromSigma Chemical (St Louis Mo.). D, L-Mox were isolated with HPLC usingthe method outlined by Ziemniak et al., 1982. D,L-Mox was taken up inwater and fractioned on a C18 column with a running buffer of 1% MeCN,pH 6.5. Column effluent was monitored at 275 nm with a UV detector. Bothisomers came out as single peaks. D Mox had a retention time of 6.7 minwhile L-Mox came out at 8.2 min. The individual isomers of Mox providedto be relatively unstable and would rapidly re-isomerize duringlyophilization making it difficult to have a reasonably pure (>98%)sample. Hence it was necessary to go directly from the HPLC to theanimal. D isomer (ca. 200 μg/ml HPLC buffer) was diluted to 50 μg/mlsaline and keep on ice until IP injection (50 μg/kg). L isomer (ca. 150μg/ml HPLC buffer) was also diluted to 50 μg/ml saline and treatedsimilarly.

Results

Two groups of eight animals each were tested for offensive aggressionfollowing treatment with 50 μg/kg D or L Mox (FIG. 9). Animals weretested 90 min after injection. D Mox significantly increased bitelatency (p<0.01) and reduced the number of bites (p<0.05). There was nosignificant difference in contact time or flank marking between the twoisomers.

SUMMARY

These data identify D moxalactam as the active isomer affectingoffensive aggressive behavior.

IX. Testing Beta-Lactam Related Antibiotics for Antiaggressive EffectsRationale

Moxalactam is chemically and pharmacologically similar to cephalosporinand penicillin antibiotics. Indeed, moxalactam is classified as acephalosporin. The basic structures of all cephalosporins and penicillinare show below. Each has a beta-lactam ring (A), in turn, cephalosporinhas a six-sided dihydrothiazine ring (B) and penicillin a five-sidedthiazolidine ring (B). These basic structures that form the chemicalnucleus for these antibiotics occur naturally in fungus. Moxalactam isnot found in nature and is characterized by an oxygen substitution forthe sulfur (S) atom in cephalosporin.

Cephalosporins and penicillin are bacteriocidal. Their antibacterialactivity is due to an inhibition of peptidoglycan synthesis in thebacterial cell walls. Although the exact mechanism of action is notfully understood, these antibiotics bind to several proteolytic enzymes,e.g., carboxypeptidases and endopeptidases, that are involved insynthesizing the peptidoglycan latticework that strengthens thebacterial cell wall. The interaction between these antibiotics and theproteolytic enzymes is reversible. It is thought that these beta-lactamantibiotics act as substrate analogs for acyl-D-alanyl-D-alanine, theendogenous substrate for these enzymes. When these bacterial enzymes arebound up with antibiotic they cannot perform their function and thebacteria lyse as they replicate.

Similar carboxypeptidases and endopeptidases are associated with cellmembranes of neurons and glia in the mammalian brain. One of their manyfunctions is to rapidly degrade neuropeptides acting asneurotransmitters. Unlike the classical neurotransmitters, e.g. dopamineand serotonin, that rely on reuptake mechanisms to stop signalactivation, neuropeptides are inactivated by their rapid degradation inthe extracellular space. These beta-lactam related antibiotics arebelieved to have psychotropic activity by interfering with themetabolism (NAALADase activity) on the numerous neuropeptides alteringthe neuropeptide milieu of the brain.

Method

Six animals were tested with equimolar concentrations (90 μM) ofMoxalactam (Mox), Ampicillin (Amp) Carbenicillin (Carb) Cefoxitin (Cef),Amoxicillin (Amox) or saline vehicle. The concentrations were adjustedto equal the 50 μg/kg dose used for MOX in previous studies. Allsolution were prepared in 0.9% NaCl and given IP. The order ofinjections was counter balanced. Animals were tested for offensiveaggression 90 min after injection (FIG. 10). There was a significantdifference between treatments on bite latency (F (5,30)=2.83; p<0.05).Both Mox and Amp significantly delayed the latency to bite (p<0.001 andp<0.05, respectively) as compared to vehicle control. There was also asignificant difference between treatments on number of bites (H=10.6;p<0.05). Both Mox and Amp drugs significantly reduced the number ofbites (p<0.05). There were no significant treatment effect on contacttime or flank marking (FIG. 11).

SUMMARY

These data indicate that the antiaggressive effect of the beta-lactamantibiotic Mox may be extended to include the beta-lactam ampicillin. Ofall of the antibiotics tested, Mox has the greatest penetrability intothe CNS. Patents given 2.0 g of Mox IV show cerebrospinal fluid levelsof drug around 30 μg/ml. The ratio of CSF to serum levels of Mox is ca.15-20%. It is estimated that the serum concentration of Mox in 140 ghamster given an IP injection of 14 μg of drug is 0.1 ng/ml. This wouldbe reflected by a CSF concentration of 15 ng/ml or brain levels of Moxapproximating 30 nM. These levels would certainly be in range tointeract effectively with neuropeptide receptors most of which havebinding affinities in the nanomolar range. Interaction with theclassical neurotransmitters would be less likely because these receptorshave Kd's in the micro and millimolar range.

Neonates with meningitis (conditions favoring CNS penetrability ofbeta-lactam antibiotics) show a ratio of CSF to serum level of Amp ofca. 10%. Cefoxitin, on the other hand has poor CNS penetrability evenwhen the meninges are inflamed. Perhaps many of the beta-lactamantibiotics would be effective in suppressing aggressive behavior andthey are simply limited by their pharmacokinetics and CNS penetrability.To test this notion it was necessary to repeat the beta-lactamantibiotic study using a higher dose of each drug.

X. High Dose Beta-Lactams

Six animals were tested with equimolar concentrations (ca. 5 mg/kg; 9mM) of Ampicillin (Amp) Carbenicillin (Carb) and Cefoxitin (Cef) orsaline vehicle. The concentrations were adjusted to equal the 5 mg/kgdose used in the dose response study for Mox. All solution were preparedin 0.9% NaCl and given IP. The order of injections was counter balanced.Animals were tested for offensive aggression 90 min after injection(FIG. 12). There was a significant difference between treatments on bitelatency (F (4,25)=5.49; p<0.01). Both Amp and Carb significantly delayedthe latency to bite (p<0.001) as compared to vehicle control. There wasalso a significant difference between treatments on number of bites(H=11.7; p<0.05). Both Amp and Carb significantly reduced the number ofbites (p<0.05 and p<0.01, respectively). There were no significanttreatment effect on contact time or flank marking (FIG. 13).

Amoxicillin was not included in this high dose beta-lactam antibioticstudy; instead, it was run in a separate study using a dose of 1 mg/kg(ca. 2 mM). Eight animals were tested for offensive aggression 90 minafter IP injection following treatment with Amox or saline vehicle (FIG.14). Each animal was given each treatment with no less than 48 hrsbetween injections. The treatments were counterbalanced. Aggressivebehavior was not significantly altered in animals treated with 1 mg/kgAmox.

SUMMARY

These data indicate that ampicillin and carbenicillin given in highenough doses can suppress offensive aggression without altering contacttime or flank marking. These data raise the possibility that thepsychotropic effect of moxalactam is shared by other beta-lactams andthat the biological mechanisms of action may be similar. Bioavailabilityand CNS penetrability, in part, may be the major component contributingto differences in biological efficacy. Indeed, more recent testingdemonstrated that clavulanic acid, a β-lactam compound having noclinically significant antibiotic activity, but a clinically importantβ-lactamase inhibition activity, exhibits a wide variety of psychotropiceffects, including antianxiety, antiaggression and cognitionenhancement, at i.p. doses less than 1 μg/kg. Its high oral absorptionand good blood brain barrier transport properties make it and relatedβ-lactamase inhibitors preferred candidates for use in the methods andthe pharmaceutical formulations in accordance with this invention.

The mechanism (s) of action for the psychotropic effects of thesebeta-lactams is now believed to be their interaction with neurogenicNAALADase. This is feasible since cephalosporins are reported to havebactericidal activity in concentrations as low as 10 nM. Note, theestimated concentration of Mox in the brain following the 50 μg/kgtreatment is ca. 30 nM.

Another possible explanation for the psychotropic activity ofbeta-lactam antibiotics is the possible blockade of knownneurotransmitter receptors or re-uptake proteins. To test this secondpossibility it was necessary to screen Mox for receptor interaction in awide range of radio ligand binding assays.

XI. Screening Moxalactam in Receptor and Transport Binding AssaysTesting Mox For Vasopressin V_(1a) And Serotonin 5ht_(1a) ReceptorInteraction

Vasopressin and serotonin are both critical neurotransmitters in thecontrol of offensive aggression in male hamsters (Ferris et al., 1998).These two neurotransmitters also are implicated in the control of humanaggression (Coccaro et al., 1998). Vasopressin facilitates aggressivebehavior while serotonin inhibits aggression, in part, by inhibiting theactivity of the vasopressin system. Blockade of vasopressin V_(1A)receptors and stimulation of serotonin 5HT_(1A) receptors in theanterior hypothalamus blocks offensive aggression (Ferris et al., 1999).Since Mox significantly suppresses offensive aggression it washypothesized it did so by interacting with either one or both of thesereceptors. To test this notion Mox was tested in a membrane bindingassay for competition for the V_(1A) receptor (Ferris et al., 1994) andin a receptor autoradiography assay for competition for the 5HT_(1A)receptors (Ferris et al., 1999). Moxalactam in a concentration of 1 μMdid not significantly displace I¹²⁵ HO-LVA (vasopressin ligand) bindingin a hamster liver membrane preparation. Similarly, Mox was ineffectivein reducing specific binding of I¹²⁵ DPAT (serotonin ligand) to tissuesections of the hamster brain.

SUMMARY

These data show that moxalactam has no direct interaction withvasopressin V_(1A) and serotonin 5HT_(1A) receptors in the hamster. Thiswould suggest that moxalactam is affecting behavior by altering theactivity of other neurochemical pathways.

Testing for Amino Acid, Adrenergic, Serotonergic, and DopaminergicReceptors And Their Transporters

Moxalactam was screened in thirty-six different binding assays byNOVASCREEN, a contract research organization based in Hanover, Md.Moxalactam was tested at 100 nM and nm in duplicate samples for each ofthe assays listed on the following page. These assays were chosenbecause their respective receptor or transporter may play a role in thepathophysiology of mental illness. Moxalactam had no significant effectin any of these binding assays.

Amino Acid Targets   Benzodiazepine, peripheral   GABA     Agonist Site    Benzodiazepine, central   GABA   Glutamate     AMPA Site     KainateSite     NMDA, Agonist Site     NMDA, Glycine [strychnine-insensitive]site   Glycine [strychnine-sensitive] site Biogenic Amine-AdrenergicTargets   Adrenergic     α_(1A)     α_(1B)     α_(2A) (human HT-29cells)     α_(2B)     α_(2C) (human recombinant)     β₁     β₂ BiogenicAmine-Serotonergic Targets   Serotonin     5HT_(1A) (human recombinant)    5HT_(1B)     5HT_(1D)     5HT_(2A) (formerly 5HT₂)     5HT_(2C)    5HT₃     5HT₄     5HT₆ (rat recombinant)     5HT₇ (rat recombinant)Biogenic Amine-Dopaminergic Targets   Dopamine     D₁     D₂ (humanrecombinant)     D₃ (rat recombinant)   Clozapine Uptake/TransporterTargets   Adrenosine   Adrenergic, Norepinephrine   Dopamine   GABA  Glutamate   Muscarinic, Choline   Serotonin Hormone Targets  Corticotropin Releasing Factor

Testing for Corticotropin Releasing Hormone Receptor

Corticotropin releasing hormone (CRH or CRF as shown on the followingpage) is a critical neurohormone in the regulation of stress. Since Moxsuppresses impulsivity, aggression, and anxiety while enhancing learningand memory it may be acting to reduce stress. For this reason, Mox wastested by NOVASCREEN in a CRF binding assay. Moxalactam at aconcentration of 100 nM had no effect in this assay.

SUMMARY

These data show that moxalactam does not interact directly with many ofthe receptors and transporters implicated in the pathophysiology ofaggression and mental illness. This leaves three possible mechanisms ofaction: 1) interaction with known receptors that were not screened,e.g., histamine, acetylcholine, and other neuropeptides, 2) interactionwith unknown or “orphan receptor,” or 3) interaction with peptidolyticenzymes (e.g., NAALADase) in the CNS that alter the chemical milieu ofthe brain.

XII. Examining Mechanism of Action Testing Peptidoglycan-PrecursorPeptide For Effects On Offensive Aggression Rationale

The beta-lactam antibiotics have a stereochemistry that resemblesacyl-D-alanyl-D-alanine, the natural substrate for the bacterialproteolytic enzymes. Presumably, this structural characteristics enablesbeta-lactam antibiotics to behave as competitive substrate blockingenzyme activity. To test this hypothesis an analog ofacyl-D-alanyl-D-alanine, peptidoglycan-precursor peptide (Nieto andPerkins 1971; Zeiger and Maurer, 1973) was tested for antiaggressiveeffects in the hamster resident/intruder paradigm.

Method

Peptidoglycan-precursor peptide, Ala-D-γ-Glu-Lys-D-Ala-D-Ala, (PPP) wasobtained from Sigma Chemical and reconstituted in DMSO and diluted in0.9% NaCl to a final concentration of ca. 2 mM. Animals wereanesthetized with sodium pentobarbital (50 mg/kg), implanted withmicroinjection guide cannulae aimed at the lateral ventricle and allowedto recover for two days before testing. On the day of testing, animals(n=6) were injected with vehicle (2% DMSO in 0.9% NaCl) or PPP in a doseof ca. 1 mg/kg in a volume of 1 μl Sixty minutes after injection,animals were retested for offensive aggression toward a smaller intruderplaced into their home cage. Two days later animals were tested againand the order of treatments reversed.

Results

Peptidoglycan-precursor peptide significantly increased the latency tobite (p<0.05) and reduced the number of bits (p<0.05) during a 10 min.Observation period (FIG. 15). There was no significant difference incontact time or flank marking between treatments (FIG. 15).

Testing Peptidoglycan-Precursor Peptide for Effects of OlfactoryDiscrimination

Six animals received an intracerebroventricular injection of vehicle or1 mg/kg PPP and tested for olfactory discrimination by measuring theirlatency to fid hidden sunflower seeds (FIG. 16). The injections werecounterbalanced with each animal receiving each treatment. Prior totesting animals were fasted for 24 hrs. Sixty min. After injectionanimals were briefly taken from their home cage while six sunflowerseeds were buried under the bedding in one corner. Animals were placedback into their home cage and scored for the latency to find the seedsin a five min. Observation period. The latency to find the seed wassignificantly (p<0.05) reduced in animals treated with PPP as comparedto vehicle.

SUMMARY

The direct injection of peptidoglycan-precursor peptide into the brainof hamsters has the same behavioral results as the peripheral injectionof Mox. Both drugs and both routes of administration significantlyreduce aggressive behavior without altering social interest of motoractivity, i.e., contact time and flank marking. In addition, theenhancement of olfactory discrimination that appears to be the simplestand most robust behavioral assay for screening beta-lactam antibioticsis similarly affected by the precursor peptide. These findings areevidence that beta-lactam antibiotics affect behavior by: 1) actingdirectly on the brain, and 2) resembling the acyl-D-alanyl-D-alaninepeptide moiety.

While clavulanic acid contains a beta-lactam ring and is structurallysimilar to penicillins and cephalosporins, it has weak antibacterialactivity with no therapeutic value as an antibiotic. However, when givenin combination with some beta-lactam antibiotics like ticarcillin(Timentin®) clavulanic acid can extend the spectrum and enhance theactivity of the antibiotic (AHFS, 1991). This synergistic activity ispossible because clavulanic acid acts as an irreversible competitiveinhibitor of bacterial beta-lactamases that naturally degrade andinactive beta-lactam antibiotics (Brown et al., 1976; Reading and Cole1977).

Clavulanic acid is commercially available in the United States but onlyin fixed combination with other drugs. Commonly prescribed Timentin® isnormally given intravenously in doses ranging from 200-300 mg/kg/day(based on ticarcillin content) which corresponds to a dose of clavulanicacid of approximately 7-10 mg/kg/day (AHFS, 1991). There are no reportedadverse reactions or contraindications for clavulanic acid given in thisdose range (Koyu et al., 1986; Yamabe et al., 1987). The data presentedbelow report clavulanic acid can alter CNS activity and behavior atdoses ranging from 10 ng to 10 μg/kg, or 1000 to 1,00,000 times lessthan used in antibacterial indications.

Clavulanic acid by itself is orally active and stable. Thebioavailability is approximately 64 to 75% (Davies et al., 1985; Boltonet al., 1986) with an elimination half-life of just under two hours.Peak plasma concentrations occur between 45 min to three hours afteringestion (Bolton et al., 1986) with a plasma half-life of over 2 hrs(Nakagawa et al., 1994). The volume of distribution is around 15 literssuggesting clavulanic acid is primarily confined to extracellular fluid(Davies et al., 1985). The CSF/plasma ratio is around 0.25, evidencethat clavulanic acid readily passes the blood-brain barrier (Nakagawa etal., 1994).

Behavioral Studies with Clavulanic Acid

I. Clavulanic Acid Dose-Response in the Seed Finding Model of Anxiety

Clavulanic acid (CLAV) is structurally similar to the beta-lactamantibiotics. A most robust and simple bioassay for screeningbeta-lactams for CNS activity is the golden hamster seed finding modelof anxiety. Briefly, hamsters are deprived of food overnight. Thefollowing day they are exposed to the additional stress of being takenfrom their home cage and placed in a novel environment for a fewminutes. This manipulation stimulates the release of the stress hormonecortisol (FIG. 37). During their absence from the home cage, sunflowerseeds are hidden under the bedding in one of the corners. When returnedto the home cage, hamsters routinely scramble along the walls for 1-2min before settling down, locating and eating the seeds. However,animals treated with the benzodiazepine anxiolytic chlordiazepoxide findseeds in less than 10 sec. This reduction in seed finding time fromminutes to seconds also occurs following treatment with moxalactam andother beta-lactam antibiotics.

Experimental Protocol

Male, Syrian golden hamsters (Mesocricetus auratus) (120-130 g) obtainedfrom Harlan Sprague-Dawley Laboratories (Indianapolis, Ind.) were housedindividually in Plexiglas cages (24 cm×24 cm×20 cm), maintained on areverse light:dark cycle (14L:10D; lights on at 19:00 hr) and providedfood and water ad libitum. A range of concentrations of CLAV (salinevehicle, 0.1, 1.0, 10, 100 1,000 ng/kg) wire tested in six groups ofhamsters (4-8/group)(FIG. 25). All tests were conducted during the darkphase of the circadian cycle under dim red illumination. Prior totesting all animals were fasted for 20-24 hrs. Ninety min afterintraperitoneal (IP) injection of drug, animals were taken from theirhome cage and placed into a holding cage for 2 min. During theirabsence, six sunflower seeds were buried under the bedding in one cornerof their home cage. Animals were placed back into their home cagerandomly facing any one of the empty corners and timed for their latencyto find the seeds in a five min observation period. Latency times wereanalyzed with a one-way ANOVA followed by Scheffe's post hoc tests.Assumption of equal variances was tested (Hartley's F-max=2.1 p>0.05)

Results

The latency to find the sunflower seeds was significantly differentbetween doses (F_((5,30))=10.0; p<0.0001). CLAV in doses of 10 ng andabove significantly (p<0.01) reduced latency times to less than 8.0 secas compared to saline vehicle with a mean latency of 104 sec. The doseof 1 ng/kg was not significantly different from vehicle control.

SUMMARY

The data show CLAV given in a dose of 10 ng/kg body weight has maximalefficacy the seed finding test. The adult male hamsters used in thesestudies weighed around 125 g. Hence, these animals were given about 1.25ng of CLAV. CLAV has a volume of distribution approximating theextracellular fluid volume. The extracellular water content of lean bodymass is approximately 22%. The concentration of 1.25 ng of CLAV in 27.5ml of water is 0.045 ng/ml or about 200 pM (formula weight of thepotassium salt of CLAV is ca. 240). Since the CSF/plasma ratio is 0.25the estimated concentration in the brain would be around 50 pM.

The seed finding model of anxiety appears to have empirical validity(McKinney 1989) i.e., drugs like benzodiazepines that are used to treatclinical anxiety are effective in the animal model. However, a widerspectrum of anxiolytics and non-effective drugs must be screened toassess the incidence of false negatives and false positive beforeadopting seed finding as a model of anxiety. Hence, it was necessary tovalidate the potential anxiolytic activity of CLAV in the traditionalelevated plus-maze.

II. Testing Clavulanic Acid in the Elevated Plus-Maze

The elevated plus-maze was developed for screening anxiolytic andanxiogenic drug effects in the rat (Pellow et al., 1985). The method hasbeen validated behaviorally, physiologically, and pharmacologically. Theplus-maze consists of two open arms and two enclosed arms. Rats willnaturally make fewer entries into the open aims than into the closedarms and will spend significantly less time in open arms. Confinement tothe open arms is associated with significantly more anxiety-relatedbehavior and higher stress hormone levels than confinement to the closedarms. Clinically effective anxiolytics, e.g., chlordiazepoxide ordiazepam, significantly increase the percentage of time spent in theopen arms and the number of entries into the open arms. Conversely,anxiogenic compounds like yohimbin or amphetamines reduce open armentries and time spent in the open arms.

Male Wistar rats weighing 250-300 g were group housed in a normal 12:12light-dark cycle with light on at 0800 hr and provided food and water adlibitum. The plus-maze consisted of two open arms, 50 cm long, 10 cmwide, with walls 40 cm high made of clear Plexiglas. The two closed armshad the same dimensions but included a roof. The Plexiglas for theclosed arms was painted black. Each pair of arms was arranged oppositeto each other to form the plus-maze. The maze was elevated to a heightof 50 cm. Eighteen animals were tested in the plus-maze 90 min followingthe IP injection of 1.0 μg/kg CLAV, 50 or vehicle control in a volume ofca. 0.3 ml. The order of treatments was counter balanced with at least48 hrs between injections. At the start of the experiment, the animalwas placed at the end of one of the open arms. Over a five minobservation period, animals were scored for the latency to enter theclosed arm, time spent in the closed arm and the number of open armentries following the first occupation of the closed arm. The studyproduced tables of repeated measures. The data between treatments werecompared with a two-way, repeated measures ANOVA followed by Bonferronipost hoc tests. There was a significant difference between treatmentsfor latency to enter the dark (F_((1,18))=8.53; p<0.01). When treatedwith CLAV (p<0.05) animals stayed in the starting open light positionlonger than when treated with vehicle (FIG. 26). The time spent in theopen arm was highly significant between treatments (F_((1,18))=144;p<0.0001) (FIG. 26). The time spent in the open arm was significantlyincreased for CLAV (p<0.01) as compared to vehicle. Finally, the openarm entries were significantly different between treatments (F (1,18)=44.0 p<0.0001) with CLAV (p<0.01) treatment showing increasedmovement into the lighted open arms as compared to vehicle (FIG. 26).

These data show CLAV given at a dose of 1 μg/kg has anxiolytic activityin the plus-maze. These data are encouraging; however, many anxiolyticssuch as the benzodiazepines depress motor activity. Since animalstreated with CLAV took a longer time to move from the lighted open armto the dark, protected, closed arm it could be argued that thisbeta-lactam did not reduce anxiety, instead it sedated the animal andretarded movement. To control for this possibility it was necessary toscreen CLAV for general motor activity in an open field paradigm.

III. Motor Activity in an Open Field Experimental Protocol

Immediately after each of the plus-maze tests reported above in SectionII, animals were tested for general motor activity in an “open field.”Animals were placed into a large clean Plexiglas cage (48×32×40 cm)devoid of bedding. This open field was delineated into equal quadrantsby tape on the underside of the cage. Animals were scored for motoractivity by counting the number of quadrants traversed in 1 min. Therewere no significant differences between CLAV and vehicle treatment onopen field activity (FIG. 27).

SUMMARY

There is no evidence in the open field test that CLAV depress motoractivity. This finding is corroborated in another behavioral study,flank marking reported in Section VII. Flank marking is a complexstereotyped motor behavior used by hamsters to disseminate pheromonesfor olfactory communication (FIG. 39). Flank marking is unaffected bytreatments with CLAV. It would appear that this beta-lactam has anadvantage over the more conventional benzodiazepine anxiolytics since itdoes not depress motor activity. However, is the anxiolytic activity ofCLAV comparable to the clinically prescribed benzodiazepines?

IV. Clavulanic Acid Vs Chlordiazepoxide in the Plus-Maze ExperimentalProtocol

Chlordiazepoxide)(Librium□ is a commonly prescribed anxiolytic that hasbeen thoroughly characterized in preclinical studies. The effectiveanxiolytic dose in the plus-maze is 10-25 mg/kg (Lister 1987; File andAranko 1988; Shumsky and Lucki 1994). In this range of doses,chlordiazepoxide (CDP) is a sedative and depresses motor activitycomplicating the interpretation of any behavioral assay that requireslocomotion (McElroy et al., 1985). However, it was discovered animalsdevelop a tolerance to the motor depression with repeated dailyadministration of CDP for several days (Shumsky and Lucki 1994). Hencein these studies, rats (n=6) were given a single IP injection of CDP (10mg/kg) each day for seven days prior to the start of the experiment.While CLAV has no effect on motor activity it was necessary to treat anequal number of rats with daily injections of CLAV (100 ng/kg) to insurea balanced experimental design. In addition there was a third group ofrats (n=6) receiving daily injections of saline vehicle. The studyreported in Section II tested CLAV at 1 μg/kg in the plus-maze. The datafrom the seed finding assay of anxiety shown in Section I suggests CLAVshould be effective between doses of 10 ng to 1 μg/kg. For this reasonCLAV was tested at 100 ng/kg in these studies.

Results

There was a significant difference between treatments (F_((2,15))=21.45,p<0.001) for the latency to enter the dark. The latency to enter thedark closed arms was significantly greater for animals treated with CLAVand CDP (p<0.01) as compared to vehicle control (FIG. 28A). There wasalso a significant difference between treatments (F_((2,15))=17.14,p<0.001) for the time spent in the light. The time spent exposed tolight in the open arms was also significantly greater for the CLAV andCDP (p<0.01) treated animals as compared to vehicle (FIG. 28A). Therewas no significant difference between treatments for open arm entries(FIG. 28B).

SUMMARY

These data show that CLAV and CDP have similar anxiolytic activity inthe elevated plus-maze. Yet, CLAV has greater potency being effective ata dose 100,000 times less than CDP. Furthermore, CLAV does not have thesedative, motor depressant activity of the conventional benzodiazepineanxiolytics. The anxiolytic effects of CLAV are immediate and do notrequire the development of tolerance to realize behavioral efficacy.However, a point of caution, benzodiazepines have another undesirableside effect for which there is no development of tolerance-amnesia(Shumsky and Lucki 1994). For example, diazepam)(Valium° selectivelyimpairs short-term memory and attention while sparing long-term memory(Liebowitz et al., 1987; Kumar et al., 1987). Hence, it was necessary totest CLAV for any untoward effects on learning and memory.

V. Clavulanic Acid and Spatial Memory in the Water Maze

The Morris water maze was developed to test spatial memory (Morris,1984). The pool is divided into quadrants usually designated North,South, East and West. The water in the pool is made opaque with milkpowder. Hidden just beneath the surface in one of the quadrants is aplatform that serves as a escape route for rodents placed into the pool.An animal is placed some where in the pool from a variety of differentstart points and is timed for latency to find the platform, percent timespent in each quadrant, distance traveled and swimming speed. Theanimals have no visual or spatial cues in the pool and must rely onextra-maze cues, i.e., objects set up outside the pool that can be seenby the swimming animal. Through a series of trials a rat develops “placelearning” or knowledge about the position of the platform based upon theextra-maze cues. The platform can be moved to a different quadrant eachday combining spatial memory with working memory. This paradigm involvesextinction of the prior memory and resolution of a new spatial problem.

Methods

The water maze consisted of a black plastic circular pool ca. 150 cm indiameter and 54 cm in height filled to a level of 35 cm with water madeopaque with powdered milk. The pool was divided into four quadrants witha platform 10 cm in diameter submerged 2 cm below the surface in thenorthwest quadrant. The water was maintained at a temperature of 25° C.Around the pool were several visual cues. Above the pool was a videocamera for tracking the movement of the experimental animal. The datacollection was completely automated using the software developed by HVSImage (Hampton, UK). Before testing, rats were familiarized with thepool and platform placed in the northwest quadrant. Each day for 4consecutive days, animals were placed into pool at random sites andgiven two min to find the platform. Animals were treated one hr beforetesting with 1.0 μg/kg CLAV (n=9) or vehicle (n=9). Following thesefamiliarization trials, animals were tested for spatial navigation. Thefirst day of testing began with the platform in the expected northwestquadrant. All behavior was videotaped for a two min observation period.After testing the animal were dried off and placed back into their homecage. On each subsequent day the platform was moved to a new quadrantand the rat started at different positions. The rat was always placedinto the pool facing the sidewall. The start positions relative to theplatform were different for each of the four trials; however, theplatform was always in the same relative position in each quadrant. Itwas positioned 20 cm in from the side of the pool and in the left cornerfrom the center facing out. The latency to find the hidden platform,path length, swim rate, and quadrant times between CLAV and vehicletreated animals were compared with a two-way, repeated measures ANOVAfollowed by Bonferroni post hoc tests.

Results

There was no main effect for drug treatment (F_((1,16))=4.17, p<0.057),days of testing (F_((3,48))=0.51, p>0.5) or interaction between factors(F_((3,48))=1.92 p>0.1) (FIG. 29) for latency to find the platform.However, animals treated with CLAV showed shorter latencies to find theplatform on Days 1 and 4 with a trend towards significance.

The strategy for finding the platform was similar for both treatments(FIGS. 30A & B) as judged by the percentage of time the animals spent ineach quadrant. For any quadrant on any day there was no significantdifference between treatments. There was a significant differencebetween days for percentage of time spent in any particular quadrant(e.g., CLAV, North Quadrant, F_((3,32))=38.81, p<0.0001). Animals spenta significant portion of their time in certain quadrants on certaindays. For example, on Day 1 both CLAV and vehicle animals spent most oftheir time in the North quadrant as compared to the other quadrants(p<0.01). This was to be expected since they had knowledge of thelocation of the platform in this quadrant from the familiarizationprocedure.

While the strategy for finding the platform as measured by percentage oftime spent in each quadrant was similar between CLAV and vehicle therewas a small but obvious difference. Animals treated with CLAV spent moretime in the correct quadrant than animals treated with vehicle. Thisdifference is particularly true on Day 2 when the CLAV animals spentover 50% (p<0.01) of their time in the correct (South) quadrant. Thevehicle animals spent less than 40% of their time in the correctquadrant, a time not significantly different from the other quadrants.By Day 4 both CLAV and vehicle spent most of their time in the correctquadrant (West). This strategy on Day 4 shows good spatial, working andprocedural memory for both treatments.

There was a significant main effect for treatment (F_((1,16))=8.40,p>0.01) on the path length to find the platform. On Day 1 CLAV treatedanimals (p<0.05) traveled a much shorter distance during the search forthe platform than vehicle animals (FIG. 31). There was no significantdifference between CLAV and vehicle on swim rate (FIG. 32).

2. Cue Navigation Method

On the day following the last day (Day 4) of spatial navigation, animalswere tested for cue navigation. In these tests, the platform was raisedabove water level. One hr before testing animals were treated with CLAVor saline vehicle. The same animals treated with CLAV during spatialnavigation were treated with CLAV for cue navigation. Animals were runthrough a series of two min trials with 45 min between trials. At eachtrial, the platform was moved to a different quadrant. The cuenavigation study was identical to the spatial navigation except theplatform was visible and the testing was done over five consecutivetrials done on a single day. Animals were scored for latency to find theplatform, percent time spent in each quadrant, path distance and swimspeed for all testing periods

Results

There was no main effect for treatments (F_((1,16))=0.553 p>0.1), trials(F_((4,64))=0.9745, p>0.1) or interaction between factors(F_((4,64))=0.7433, p>0.5) for latency to find the platform during cuenavigation (FIG. 33).

As in spatial navigation, the strategy for finding the platform was verysimilar for both treatments (FIGS. 34A & B) as judged by the percentageof time the animals spent in each quadrant. For any quadrant on anytrial there was no significant difference between treatments (e.g.,Trial 1, North, F_((1,16))=0.099, p>0.5). There was a significantdifference for percentage of time spent in any particular quadrant foreither treatment for most of the trials, most notably for CLAV.

The distance traveled to find the platform was not significantlydifferent between CLAV and vehicle animals (F_((1,16))=0.23 p>0.5) (FIG.35). While there was no significant main effect for treatment on swimrate (F_((1,16))=0.926, p>0.1), there was a significant trails effect(F_((4,64))=7.87, p<0.001) and interaction between factors(F_((4,64))=2.56, p<0.05). Both treatments, but particularly CLAV showedreduced swim rates by Trial 4 (p<0.01) and Trial 5 (p<0.05). Thisprobably reflects the fact that they knew where to look for the platformas shown in FIGS. 34A & B.

SUMMARY

Clavulanic acid treated animals do not show any loss in learning andmemory when tested for spatial and cue navigation in the Morris watermaze. Indeed, on distance traveled to the hidden platforms andpercentage of time spent in the correct quadrant for both spatial andcue navigation, CLAV treated animals showed better performance thanvehicle. These data show that the anxiolytic profile of CLAV is notaccompanied by any disruption in learning and memory as is the case withbenzodiazepine anxiolytics.

VI. Clavulanic Acid and the Stress Response

The ability of CLAV to reduce anxiety in stressful situations, i.e. thefood deprivation and novel environment in the seed finding assay, andexposure to light and a novel environment in the elevated plus-maze,without altering motor activity or cognitive function is a significantfinding. The potential of CLAV as an anxiolytic and therapeutic in thetreatment of numerous affective disorders could be broadened if we had aclearer understanding of its mechanism of action. For example, couldCLAV be altering anxiety by suppressing the natural stress response? Thecommonly prescribed benzodiazepine anxiolytics block both the normalcircadian release and stress-mediated release of the hormone cortisol(Gram and Christensen, 1986; Petraglia et al., 1986; Hommer et al.,1986).

The simple procedure of placing an adult male hamster into a novelenvironment for 5 min causes a significant, predictable increase inblood levels of cortisol (Weinberg and Wong 1986). This novelty test wasused to assess the effects of CLAV on stress-induced release ofcortisol. Two groups of male hamsters were treated IP with either CLAV(10 μg/kg, n=6), or saline vehicle (n=4). A third group (n=4) receivedno treatment or isolation stress and served as a control for basallevels of cortisol. Sixty min after treatment animals were taken fromtheir home cage and placed into a novel cage for 5 min. Afterwardsanimals were sacrificed by decapitation and trunk blood collected forradioimmunoassay of cortisol. All animals were tested under reverselight:dark conditions four hrs into the dark cycle. Data were comparedwith a one-way ANOVA followed by Fisher PLSD post hoc tests.

There was a significant difference in the stress release of cortisolbetween treatments (F_((2,11))=10.03 p<0.01). Vehicle (p<0.05) and CLAV(p<0.01) showed more than twice the blood level of cortisol as comparedto the untreated, non-stressed control (FIG. 37).

The data show that the beta-lactam anxiolytic CLAV has no ostensibleeffect on the release of cortisol in response to the mild stress ofexposure to a novel environment. This detail, combined with the absenceof motor depression and cognitive impairment makes CLAV unique amongstthe anxiolytics and suggests a highly specific, novel mechanism ofaction. At first glance one might think it would be advantageous tosuppress the stress response. Indeed, hypercortisolism has beenimplicated in the pathophysiology of depression (Sacher et al., 1973).Chronic psychosocial stress leading to dysfunctional, hyperactiveadrenal glands can be life threatening. However, a responsivehypothalamic-pituitary-adrenal axis is critical for normal physiologyand behavior. Stressors that would normally help animals adapt to theenvironment can be fatal without the appropriate release of cortisol.

VII. Territorial or Offensive Aggression

Continuing to study the CNS activity of CLAV in more complex behavioralmodels may help to clarify its mechanism(s) of action. For example,antagonistic, social interactions between animals require riskassessment, communicative and agonistic behaviors to settle disputesover territory, mates, food, etc. The neurotransmitters serotonin andvasopressin are fundamental in the CNS organization and expression ofthese behaviors in animals and humans (Ferris et al., 1997; Coccaro etal., 1998; Ferris 2000). To this end, CLAV was tested for effects onterritorial or offensive aggression, i.e. defense of the home burrowagainst intruders.

Agonistic behavior can be classified as either offensive or defensiveaggression (Blanchard and Blanchard, 1977; Adams, 19798; Albert andWalsh, 1984). Offensive aggression is characterized by an aggressorinitiating an attack on an opponent; while, defensive aggression lacksactive approach. Both types of aggression have their own uniqueneurobehavioral systems. The stimuli that elicit offensive and defenseattack are different, as are the sequences of behaviors that accompanyeach agonistic response. While much of the empirical data supporting thenotion of unique offensive and defensive neural networks have beencollected from animal models, there are interesting and compellingsimilarities in human aggression that suggest a similar neuralorganization (Blanchard, 1984). Offensive aggression is easily studiedusing male golden hamsters tested in a resident/intruder paradigm, anestablished model of offensive aggression (Ferris and Potegal 1988) inthe context of defending the home burrow. Placing an unfamiliar malehamster into the home cage of another male hamster elicits awell-defined sequence of agonistic behaviors from the resident thatincludes offensive aggression. Hamsters are nocturnal and as such allbehavioral tests were performed during the first four hrs of the darkphase under dim red illumination. The resident was scored for offensiveaggression, e.g., latency to bite the intruder, the total number ofbites, total contact time with the intruder and flank marking over a 10min test period (Ferris and Potegal, 1988). Flank marking is a form ofolfactory communication in which a hamsters arches its back and rubspheromone producing flank glands against objects in the environment(Johnston, 1986). Flank marking frequency is greatly enhanced duringaggressive encounters and is particularly robust in dominant animalsinitiating and winning fights (Ferris et al., 1987).

Five male golden hamsters (130-140 g) were given IP injections of CLAV(200 μg/kg) and saline vehicle in a volume of ca. 0.2 ml. In pilotstudies, it was discovered CLAV given IP at 1.0 μg/kg had no effect onaggressive behavior. Hence, it was necessary to test CLAV at a higherconcentration but in a dose range that was still acceptable forpharmaceutical studies on aggressive behavior. Vehicle and CLAVtreatments were counter balanced and randomized so all five animalsreceived each treatment separated by at least 48 hrs. Animals weretested 90 min after treatment over a 10 min observation period.Latencies and contact time were analyzed with a two-way ANOVA.Non-parametric data, i.e., number of bites and flank marks were analyzedby Wilcoxon matched-pairs signed-ranks test.

While there was no significantmain effect for drug treatment(F_((1,3))=7.40, P<0.07) for latency to bite the intruder there was atrend toward significance (FIG. 38). There was no significant maineffect for drug treatment (F_((1,3))=2.85, p>0.1) on contact time withthe intruder (FIG. 38). There was a significant difference between drugtreatments (T=3.0, p<0.05, N=8) and the number of bites on the intruder.CLAV treatment reduced the median number of bites to six as compared tothirteen for vehicle treated animals (FIG. 39). There was no significanteffect of drug treatment (T=4.0, p>0.1, N=5) on the resident's flankmarking behavior (FIG. 39).

Clavulanic acid hasmodestantiaggressive or serenic-like properties.Serenics are drugs used to treat impulsivity and violence (Olivier andMos, 1991). Serenics should suppress offensive aggression withoutinterfering with social, appetitive and cognitive behaviors. Socialinterest in an intruder, i.e. contact time was not altered by CLAV.Development of eltoprazine, one of the first serenics, was abandoned, inpart, because it was found to increase fear and anxiety in animals(Olivier et al., 1994). The potent anxiolytic activity of CLAV excludesthis possibility.

VIII. Interactions with Glutamyl Carboxypeptidase

CLAV has a very high binding affinity for the beta-lactamases. It ishypothesized that the presence of mammalian homologies to thesebacterial enzymes and that these homologous proteins are involved in theregulation of neurotransmitter levels in the CNS. E Coli TEM betalactamase has been cloned sequenced and crystilized to determine theactive site motifs. The four putative binding sites on beta lactamasethat could accommodate CLAV are designated active site I, II, III, andIV. These active sites, sequence location, and amino acid (AA) sequencesare as follows:

Site I: 35 AA's downstream from N-terminus: STTK (SEQ ID NO: 1) Site II:57 AA's downstream from STTK (SEQ ID NO: 1) motif: SGC, SGN, or SAN SiteIII: 111 AA's downstream from SGC motif: KTG Site IV: 41 AA's downstreamfrom SGC motif: ENKD (SEQ ID NO: 2)

Screening for amino acid sequence homologies between thesebeta-lactamase binding sites and mammalian enzymes, Revaax scientistsidentified an enzyme system in the brain that CLAV would potentiallybind in a similar manner to beta-lactamase. The enzyme glutamylcarboxypeptidase (N-acetyl, alpha linked, acidic dipeptidase) orNAALADase (Pangalos et al, 1999) is responsible for regulating theglutamatergic neurotransmission pathways whose effects would beexpressed in such behavioral outcomes as aggression, memory/cognition,and anxiety. As a result of the almost perfect overlap of the putativeactive sites of beta-lactamase and the conserved sequences in human andrat NAALADase, it was hypothesized that CLAV affects behavior byinhibiting NAALADase activity. The overlap sequence similarity betweenbeta-lactamase and NAALADase as shown below:

Site I: Beta-lactamase: 35 AA's downstream from N-terminus: STTK (SEQ IDNO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID NO:3)Site II: Beta-lactamase: 57 AA's downstream from STTK (SEQ ID NO: 1)motif: SGC, SGN, or SAN NAALADase: 59 AA's downstream from STQK (SEQ IDNO: 3) motif: SFG Site III: Beta-lactamase: 111 AA's downstream from SGCmotif: KTG NAALADase: 110 AA's downstream from SFG motif: KLG Site IV:Beta-lactamase: 41 AA's downstream from SGC motif: ENKD (SEQ ID NO: 2)NAALADase: 41 AA's downstream from SFG motif: ERGV (SEQ ID NO: 4)

Clavulanic acid inhibits gram negative beta-lactamase enzymes in therange of 15-34 nM. CLAV is effective at a dose of 10 ng/kg in the seedfinding model of anxiety (pg 3). If NAALADase were the human homologueto beta-lactamase, then CLAV would be predicted to be a high affinitysubstrate.

IX. Seed Finding Following Blockade of Naaladase Activity

It was hypothesized that CLAV functioned as an anxiolytic in the seedfinding assay by blocking NAALADase activity in the brain. If thisnotion were true then it would be predicted that drugs known to blockNAALADase should also enhance seed finding. To this end, animals weretreated with N-acetyl-beta-aspartyl-glutamic acid (beta-NAAG), acompetitive inhibitor of NAALADase (Serval et al., 1992) and tested inthe seed finding model of anxiety. The study was similar to thatoutlined in Section I with one notable exception. Since beta-NAAG doesnot readily cross the blood-brain barrier, it had to be injecteddirectly into the lateral ventricle where it could be carried bycerebrospinal fluid throughout the brain via the ventricular system.Beta-NAAG (FW 304) was given in a dose of 3 ng in a volume of 1 μlsaline ICV. The average adult hamster brain weights ca. 1.2 g of which22% is extracellular fluid. The estimated beta-NAAG concentration was 11ng/ml or 36 nM.

Two groups of six animals each were fasted overnight as previouslydescribed and tested the following day. One group was treated withbeta-NAAG and the other saline vehicle and one hour later timed forlatency to find the hidden sunflower seeds. A Student t-test forunpaired data was used for statistical comparisons.

The difference in latency to find the seeds was significantly (p<0.001)different between treatments (FIG. 40). Indeed, the none of the sixanimals microinjected with saline vehicle found the seeds in the fiveminute observation period. However, three days later when these sameanimals were microinjected with beta-NAAG (3 ng/μl) and tested for seedfinding they showed a mean latency of 21.8±9.7 sec. The data show thatbeta-NAAG a specific NAALADase inhibitor, can dramatically reduce thelatency to find hidden sunflower seeds, a biological activity shared byCLAV. Since beta-NAAG was active in the seed finding model of anxiety,then the hypothesis that beta-NAAG and CLAV share a common mechanism ofaction is not rejected. From these data the hypothesis can be expandedto predict that beta-NAAG and CLAV show similar effects on a range ofbiological and behavioral measures. To this end, animals were tested foroffensive aggression in the resident intruder paradigm as described inSection X. As reported earlier, when given in high concentrations, CLAVhas only a modest effect on offensive aggression. While CLAV can enhanceseed finding at a dose of 10 ngkg it has only a modest effect onoffensive aggression even with doses as high as 200 μg/kg. If beta-NAAGand CLAY share a common mechanism then beta-NAAG should have little orno effect on aggression.

X. Effect of NAALADase Blockade on Offensive Aggression

The animals tested in this study were those used in Section IX. Afterthe seed finding assay, beta-NAAG (n=6) and saline vehicle (n=6) treatedanimals remained in their home cage and were presented with a smaller,male intruder. The resident was scored for latency to bite, bites,contact time and flank marking over a 10 min observation period. Latencyto bite and contact time between treatments were compared with Studentt-tests. Non-parametric measures of bites and flank marks for beta-NAAGvs vehicle were compared with Mann-Whitney.

There were no significant differences between beta-NAAG andvehicle-treated animals for any measures of offensive aggression (FIGS.41 & 42).

Blocking NAALADase activity with beta-NAAG does not alter offensiveaggression as tested in the resident intruder paradigm. This finding isnot inconsistent with the notion that CLAV and beta-NAAG share a commonmechanism-blockade of NAALADase activity.

1. A neurotherapeutic pharmaceutical composition in unit dosage formcomprising a neurotherapeutically effective amount of a carboxypeptidaseE inhibitor and a pharmaceutically acceptable carrier thereof; providingthat when the carboxypeptidase E inhibitor is a β-lactam antibiotic, theneurotherapeutically effective amount of the carboxypeptidase Einhibitor is less than an antibiotically effective amount of thecarboxypeptidase E inhibitor if such β-lactam antibiotic were to beadministered in a unit dosage form by the same route of administration.2. The neurotherapeutic pharmaceutical composition of claim 1 whereinthe neurotherapeutically effective amount is an amount effective fortreating behavioral disorders or enhancing cognitive function inpatients in need of such therapy.
 3. The neurotherapeutic pharmaceuticalcomposition of claim 1 wherein the carboxypeptidase E inhibitor is acompound comprising a β-lactam ring structure.
 4. The neurotherapeuticpharmaceutical composition of claim 1 wherein the carboxypeptidase Einhibitor is selected from the group consisting of penams, cephems,1-oxa-1-dethia cephems, clavams, clavems, azetidinones, carbapenams,carbapenems and carbacephems.
 5. The neurotherapeutic pharmaceuticalcomposition of claim 1 wherein the carboxypeptidase E inhibitor is apenam or cephem compound.
 6. The neurotherapeutic pharmaceuticalcomposition of claim 1 wherein the carboxypeptidase E inhibitor is acephem sulfoxide or cephem sulfone compound.
 7. The neurotherapeuticpharmaceutical composition of claim 1 wherein the carboxypeptidase Einhibitor is a cephem sulfoxide compound.
 8. The neurotherapeuticpharmaceutical composition of claim 1 wherein the carboxypeptidase Einhibitor is a cephem sulfone compound.
 9. The neurotherapeuticpharmaceutical composition of claim 1 wherein the carboxypeptidase Einhibitor is a penam sulfoxide or penam sulfone compound.
 10. Theneurotherapeutic pharmaceutical composition of claim 1 wherein thecarboxypeptidase E inhibitor is a penam sulfone compound.
 11. Theneurotherapeutic pharmaceutical composition of claim 1 wherein thecarboxypeptidase E inhibitor is a penam sulfoxide compound.
 12. Theneurotherapeutic pharmaceutical composition of claim 1 wherein thecarboxypeptidase E inhibitor is a penicillin or cephalosporin.
 13. Theneurotherapeutic pharmaceutical composition of claim 1 wherein thecarboxypeptidase E inhibitor is a sulfoxide or sulfone derivative of apenicillin or of a cephalosporin.
 14. The neurotherapeuticpharmaceutical composition of claim 1 wherein the carboxypeptidase Einhibitor is a 1-dethia-1-oxa-cephem compound.
 15. The neurotherapeuticpharmaceutical composition of claim 1 wherein the carboxypeptidase Einhibitor is a compound of the formula

wherein R is a salt forming group or an active ester forming group; R₁is hydrogen or C₁-C₄ alkoxy; X is S, SO, SO₂, O, or CH₂; and T is C₁-C₄alkyl, halo, hydroxy, O(C₁-C₄) alkyl, or —CH₂B wherein B is the residueof a nucleophile B:H.
 16. The neurotherapeutic pharmaceuticalcomposition of claim 1 wherein the carboxypeptidase E inhibitor ismoxalactam or a pharmaceutically acceptable salt or ester thereof.